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  • 1.
    Abramenkovs, Andris
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Stenerlöw, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Measurement of DNA-Dependent Protein Kinase Phosphorylation Using Flow Cytometry Provides a Reliable Estimate of DNA Repair Capacity2017In: Radiation Research, ISSN 0033-7587, E-ISSN 1938-5404, Vol. 188, no 6, p. 597-604Article in journal (Refereed)
    Abstract [en]

    Uncontrolled generation of DNA double-strand breaks (DSBs) in cells is regarded as a highly toxic event that threatens cell survival. Radiation-induced DNA DSBs are commonly measured by pulsed-field gel electrophoresis, microscopic evaluation of accumulating DNA damage response proteins (e.g., 53BP1 or gamma-H2AX) or flow cytometric analysis of gamma-H2AX. The advantage of flow cytometric analysis is that DSB formation and repair can be studied in relationship to cell cycle phase or expression of other proteins. However, gamma-H2AX is not able to monitor repair kinetics within the first 60 min postirradiation, a period when most DSBs undergo repair. A key protein in non-homologous end joining repair is the catalytic subunit of DNA-dependent protein kinase. Among several phosphorylation sites of DNA-dependent protein kinase, the threonine at position 2609 (T2609), which is phosphorylated by ataxia telangiectasia mutated (ATM) or DNA-dependent protein kinase catalytic subunit itself, activates the end processing of DSB. Using flow cytometry, we show here that phosphorylation at T2609 is faster in response to DSBs than gamma-H2AX. Furthermore, flow cytometric analysis of T2609 resulted in a better representation of fast repair kinetics than analysis of gamma-H2AX. In cells with reduced ligase IV activity, and wild-type cells where DNA-dependent protein kinase activity was inhibited, the reduced DSB repair capacity was observed by T2609 evaluation using flow cytometry. In conclusion, flow cytometric evaluation of DNA-dependent protein kinase T2609 can be used as a marker for early DSB repair and gives a better representation of early repair events than analysis of gamma-H2AX.

  • 2.
    Adler, Jeremy
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Quantification of Colocalisation; Co-Occurrence, Correlation, Empty Voxels, Regions of Interest and Thresholding2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 602A-602AArticle in journal (Other academic)
    Abstract [en]

    Measuring colocalisation is not straightforward with a plethora of coefficients that encapsulate different definitions. Measurements may also be implemented differently. Not only do measurements differ; interconversion is impossible making comparisons challenging. There is a need to cull coefficients and for clear definitions of what precisely is meant by colocalisation in individual studies. Colocalisation can be considered to have two components; co-occurrence which reports whether the fluorophores are found together and correlation which reports on the similarity in their patterns of intensity.

  • 3.
    Adler, Jeremy
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sintorn, Ida-Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Strand, Robin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Conventional analysis of movement on non-flat surfaces like the plasma membrane makes Brownian motion appear anomalous2019In: Communications Biology, E-ISSN 2399-3642, Vol. 2, article id 12Article in journal (Refereed)
  • 4. Alizadehheidari, Mohammadreza
    et al.
    Werner, Erik
    Noble, Charleston
    Nyberg, Lena
    Fritzsche, Joachim
    Mehlig, Bernhard
    Tegenfeldt, Jonas
    Ambjoernsson, Tobias
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Westerlund, Fredrik
    Nanoconfined Circular DNA2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 274A-274AArticle in journal (Other academic)
    Abstract [en]

    Nanofluidic channels have become a versatile tool to manipulate single DNA molecules. They allow investigation of confined single DNA molecules from a fundamental polymer physics perspective as well as for example in DNA barcoding techniques.

  • 5. Allahverdiyeva, Yagut
    et al.
    Mamedov, Fikret
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
    Mäenpää, Pirkko
    Vass, Imre
    Aro, Eva-Mari
    Modulation of photosynthetic electron transport in the absence of terminal electron acceptors: characterization of the rbcL deletion mutant of tobacco2005In: Biochimica et Biophysica Acta (BBA) - Bioenergetics, ISSN 0005-2728, Vol. 1709, no 1, p. 69-83Article in journal (Refereed)
  • 6.
    Allison, Timothy M.
    et al.
    Biomolecular Interaction Centre, School of Physical and Chemical Sciences, University of Canterbury, Christchurch, New Zealand.
    Degiacomi, Matteo T.
    Department of Physics, Durham University, Durham, UK.
    Marklund, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Jovine, Luca
    Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.
    Elofsson, Arne
    Science for Life Laboratory and Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden.
    Benesch, Justin L. P.
    Department of Chemistry, University of Oxford, Oxford, UK.
    Landreh, Michael
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet – Biomedicum, Stockholm, Sweden.
    Complementing machine learning‐based structure predictions with native mass spectrometry2022In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 31, no 6, article id e4333Article in journal (Refereed)
    Abstract [en]

    The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.

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  • 7.
    Almaqwashi, Ali A.
    et al.
    Northeastern Univ, Dept Phys, Boston, MA 02115 USA..
    Andersson, Johanna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. Chalmers, Dept Chem & Chem Engn, S-41296 Gothenburg, Sweden..
    Lincoln, Per
    Chalmers, Dept Chem & Chem Engn, S-41296 Gothenburg, Sweden..
    Rouzina, Ioulia
    Ohio State Univ, Dept Chem & Biochem, Columbus, OH 43210 USA..
    Westerlund, Fredrik
    Chalmers, Dept Biol & Biol Engn, S-41296 Gothenburg, Sweden..
    Williams, Mark C.
    Northeastern Univ, Dept Phys, Boston, MA 02115 USA..
    Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 6, p. 1255-1263Article in journal (Refereed)
    Abstract [en]

    DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [mu-dppzip(phen)(4)Ru-2](4+) (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)(2)dppz) and the distal subunit (Ru(phen)(2)ip), respectively, each of which can be either right-handed (Delta) or left-handed (Lambda). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Delta x(eq), and the dynamic DNA structural deformations during ligand association x(on) and dissociation x(off). We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a K-d of 27 +/- 3 nM for (Delta,Lambda)-Piz to a K-d of 622 +/- 55 nM for (Lambda,Delta)-Piz. The striking affinity decrease is correlated with increasing Delta x(eq) from 0.30 +/- 0.02 to 0.48 +/- 0.02 nm and x(on) from 0.25 +/- 0.01 to 0.46 +/- 0.02 nm, but limited x(off) changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics.

  • 8.
    Amselem, Elias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Kipper, Kalle
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Real- Time Single Protein Tracking with Polarization Readout using a Confocal Microscope2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 295A-295AArticle in journal (Other academic)
  • 9.
    Andersson, Sara B. E.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Frenning, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Alderborn, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gråsjö, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Effect of fluid velocity and particle size on the hydrodynamic diffusion layer thickness2022In: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 180, p. 1-10Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to determine the thickness of the hydrodynamic diffusion layer (h(HDL)) of three poor water-soluble compounds under laminar fluid flow using a single particle dissolution technique. The single particle dissolution experiments were performed in a flowing aqueous medium using four different fluid velocities (v), ranging from 46 to 103 mm/s. The particles used had an initial radius (r) of 18.8 to 52.3 mu m. The determined h(HDL) values were calculated from both dissolution experiments and computational fluid dynamics (CFD) simulation. In this study, single particle dissolution experiments gave, with one exception, h(HDL) values in the range of 2.09 to 8.85 mu m and corresponding simulations gave h(HDL) values in the range of 2.53 to 4.38 mu m. Hence, we found a semi-quantitative concordance between experimental and simulated determined h(HDL) values. Also, a theoretical relation between the dependence of hHDL on particle radius and flow velocity of the medium was established by a series of CFD simulations in a fluid velocity range of 10-100 mm/s and particle size (radius) range of 5-40 mu m. The outcome suggests a power law relation of the form h(HDL)alpha r(3/5)v(-2/5). In addition, the h(HDL) seems to be independent of the solubility, while it has a diffusion coefficient dependence. In conclusion, the hHDL values were determined under well-defined conditions; hence, this approach can be used to estimate the h(HDL) under different conditions to increase the understanding of the mass transfer mechanisms during the dissolution process.

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  • 10. Aquila, Andrew
    et al.
    Hunter, Mark S.
    Doak, R. Bruce
    Kirian, Richard A.
    Fromme, Petra
    White, Thomas A.
    Andreasson, Jakob
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Arnlund, David
    Bajt, Saša
    Barends, Thomas R. M.
    Barthelmess, Miriam
    Bogan, Michael J.
    Bostedt, Christoph
    Bottin, Hervé
    Bozek, John D.
    Caleman, Carl
    Coppola, Nicola
    Davidsson, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry.
    DePonte, Daniel P.
    Elser, Veit
    Epp, Sascha W.
    Erk, Benjamin
    Fleckenstein, Holger
    Foucar, Lutz
    Frank, Matthias
    Fromme, Raimund
    Graafsma, Heinz
    Grotjohann, Ingo
    Gumprecht, Lars
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hampton, Christina Y.
    Hartmann, Andreas
    Hartmann, Robert
    Hau-Riege, Stefan
    Hauser, Günter
    Hirsemann, Helmut
    Holl, Peter
    Holton, James M.
    Hömke, André
    Johansson, Linda
    Kimmel, Nils
    Kassemeyer, Stephan
    Krasniqi, Faton
    Kühnel, Kai-Uwe
    Liang, Mengning
    Lomb, Lukas
    Malmerberg, Erik
    Marchesini, Stefano
    Martin, Andrew V.
    Maia, Filipe R.N.C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Messerschmidt, Marc
    Nass, Karol
    Reich, Christian
    Neutze, Richard
    Rolles, Daniel
    Rudek, Benedikt
    Rudenko, Artem
    Schlichting, Ilme
    Schmidt, Carlo
    Schmidt, Kevin E.
    Schulz, Joachim
    Seibert, M. Marvin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Soltau, Heike
    Shoeman, Robert L.
    Sierra, Raymond
    Starodub, Dmitri
    Stellato, Francesco
    Stern, Stephan
    Strüder, Lothar
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Ullrich, Joachim
    Wang, Xiaoyu
    Williams, Garth J.
    Weidenspointner, Georg
    Weierstall, Uwe
    Wunderer, Cornelia
    Barty, Anton
    Spence, John C. H.
    Chapman, Henry N.
    Time-resolved protein nanocrystallography using an X-ray free-electron laser2012In: Optics Express, E-ISSN 1094-4087, Vol. 20, no 3, p. 2706-2716Article in journal (Refereed)
    Abstract [en]

    We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.

  • 11.
    Arner, P.
    et al.
    Karolinska Inst, Karolinska Univ Hosp, Dept Med, Stockholm, Sweden.
    Bernard, S.
    Univ Lyon, CNRS, UMR5208, Inst Camille Jordan, Villeurbanne, France.
    Appelsved, L.
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.
    Fu, K. -Y
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    Andersson, D. P.
    Karolinska Inst, Karolinska Univ Hosp, Dept Med, Stockholm, Sweden.
    Salehpour, Mehran
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Applied Nuclear Physics.
    Thorell, A.
    Karolinska Inst, Danderyd Hosp, Dept Clin Sci, Stockholm, Sweden;Karolinska Inst, Ersta Hosp, Dept Surg, Stockholm, Sweden.
    Ryden, M.
    Karolinska Inst, Karolinska Univ Hosp, Dept Med, Stockholm, Sweden.
    Spalding, K. L.
    Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden;Integrated Cardio Metab Ctr, Dept Med, Stockholm, Sweden.
    Adipose lipid turnover and long-term changes in body weight2019In: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 25, no 9, p. 1385-1389Article in journal (Refereed)
    Abstract [en]

    The worldwide obesity epidemic(1) makes it important to understand how lipid turnover (the capacity to store and remove lipids) regulates adipose tissue mass. Cross-sectional studies have shown that excess body fat is associated with decreased adipose lipid removal rates(2,3). Whether lipid turnover is constant over the life span or changes during long-term weight increase or loss is unknown. We determined the turnover of fat cell lipids in adults followed for up to 16 years, by measuring the incorporation of nuclear bomb test-derived C-14 in adipose tissue triglycerides. Lipid removal rate decreases during aging, with a failure to reciprocally adjust the rate of lipid uptake resulting in weight gain. Substantial weight loss is not driven by changes in lipid removal but by the rate of lipid uptake in adipose tissue. Furthermore, individuals with a low baseline lipid removal rate are more likely to remain weight-stable after weight loss. Therefore, lipid turnover adaptation might be important for maintaining pronounced weight loss. Together these findings identify adipose lipid turnover as an important factor for the long-term development of overweight/obesity and weight loss maintenance in humans.

  • 12.
    Ashrafuzzaman, Md
    et al.
    King Saud Univ, Coll Sci, Dept Biochem, Riyadh 11451, Saudi Arabia..
    Khan, Zahid
    King Saud Univ, Coll Sci, Dept Biochem, Riyadh 11451, Saudi Arabia..
    Alqarni, Ashwaq
    King Saud Univ, Coll Sci, Dept Biochem, Riyadh 11451, Saudi Arabia..
    Alanazi, Mohammad
    King Saud Univ, Coll Sci, Dept Biochem, Riyadh 11451, Saudi Arabia..
    Alam, Mohammad Shahabul
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering. King Saud Univ, Coll Engn, Dept Chem Engn, Riyadh 11451, Saudi Arabia.;Uppsala Univ, Dept Engn Sci, Appl Mat Sci, S-75103 Uppsala, Sweden..
    Cell Surface Binding and Lipid Interactions behind Chemotherapy-Drug-Induced Ion Pore Formation in Membranes2021In: Membranes, E-ISSN 2077-0375, Vol. 11, no 7, article id 501Article in journal (Refereed)
    Abstract [en]

    Chemotherapy drugs (CDs) disrupt the lipid membrane's insulation properties by inducing stable ion pores across bilayer membranes. The underlying molecular mechanisms behind pore formation have been revealed in this study using several methods that confirm molecular interactions and detect associated energetics of drugs on the cell surface in general and in lipid bilayers in particular. Liposome adsorption and cell surface binding of CD colchicine has been demonstrated experimentally. Buffer dissolved CDs were considerably adsorbed in the incubated phospholipid liposomes, measured using the patented 'direct detection method'. The drug adsorption process is regulated by the membrane environment, demonstrated in cholesterol-containing liposomes. We then detailed the phenomenology and energetics of the low nanoscale dimension cell surface (membrane) drug distribution, using atomic force microscopy (AFM) imaging what addresses the surface morphology and measures adhesion force (reducible to adhesive energy). Liposome adsorption and cell surface binding data helped model the cell surface drug distribution. The underlying molecular interactions behind surface binding energetics of drugs have been addressed in silico numerical computations (NCs) utilizing the screened Coulomb interactions among charges in a drug-drug/lipid cluster. Molecular dynamics (MD) simulations of the CD-lipid complexes detected primarily important CD-lipid electrostatic and van der Waals (vdW) interaction energies. From the energetics point of view, both liposome and cell surface membrane adsorption of drugs are therefore obvious findings. Colchicine treated cell surface AFM images provide a few important phenomenological conclusions, such as drugs bind generally with the cell surface, bind independently as well as in clusters of various sizes in random cell surface locations. The related adhesion energy decreases with increasing drug cluster size before saturating for larger clusters. MD simulation detected electrostatic and vdW and NC-derived charge-based interactions explain molecularly of the cause of cell surface binding of drugs. The membrane binding/association of drugs may help create drug-lipid complexes with specific energetics and statistically lead to the creation of ion channels. We reveal here crucial molecular understanding and features of the pore formation inside lipid membranes that may be applied universally for most of the pore-forming existing agents and novel candidate drugs.

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  • 13.
    Ashrafzadeh, Parham
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Dinic, Jelena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Actin Filaments Attachment to the Plasma Membrane Cause the Formation of Ordered Lipid Domains in Live Cells2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 706A-706AArticle in journal (Other academic)
    Abstract [en]

    The aim of this study was to investigate the relationship between ordered plasma membrane nanodomains and actin filaments using di-4-ANEPPDHQ and laurdan together with the reagents that affect actin filament dynamics in live Jurkat and primary T cells. The degree of lipid packing can be quantified using polarity sensitive membrane dyes such as laurdan and di-4-ANEPPDHQ. These two dyes display a red shift in their emission peaks for membranes in ld phase relative to lo phase. Laurdan is uncharged and can easily flip between two leaflets of the plasma membrane and we demonstrate that it reports equally on the two leaflets of the plasma membrane.

  • 14.
    Ashrafzadeh, Parham
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Parmryd, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Methods applicable to membrane nanodomain studies?2015In: Essays in Biochemistry, ISSN 0071-1365, E-ISSN 1744-1358, Vol. 57, p. 57-68Article, review/survey (Refereed)
    Abstract [en]

    Membrane nanodomains are dynamic liquid entities surrounded by another type of dynamic liquid. Diffusion can take place inside, around and in and out of the domains, and membrane components therefore continuously shift between domains and their surroundings. In the plasma membrane, there is the further complexity of links between membrane lipids and proteins both to the extracellular matrix and to intracellular proteins such as actin filaments. In addition, new membrane components are continuously delivered and old ones removed. On top of this, cells move. Taking all of this into account imposes great methodological challenges, and in the present chapter we discuss some methods that are currently used for membrane nanodomain studies, what information they can provide and their weaknesses.

  • 15.
    Ayranci, Diyar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Design, expression and purification of virus-like particles derived from metagenomic studies: Virus-like Particles (VLP) of novel Partitiviridae species, Hubei.PLV 11, and novel Soutern pygmy squid flavilike virus were designed, expressed using the bac-to-bac expression system and then pruified using various methods2021Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    Viruses are entities which are made of a few genes and are reliant on obligate parasitism to propagate. Due to the obligate connection to their hosts, virus evolution is constrained to the type of host. Viruses however do transmit to evolutionary distinct hosts; in these cases, the phylogenetic relationship of the hosts usually are close. In some instances, RNA-viruses have made host jumps between evolutionary distant hosts, such as the host jump from invertebrates to vertebrates, and fungi to arthropod. Partitiviruses are double stranded RNA viruses which mainly infect fungi and plants. The defining characteristic of these double stranded RNA viruses are the double layered capsids which are formed by a single open reading frame (ORF). The capsid proteins form icosahedral virus particles which are in the magnitude of 30-40 nm. Metagenomic studies have discovered partitiviruses originating from an insect in the Odanata family, a finding which contradicts the fungal host specificity of partitiviruses. The finding of the Hubei.PLV 11 thus implies the existence of a partitiviruses containing structural elements in their capsids which could be involved in the infection of arthropods. Thus, this virus could be used as a model for a structural comparison with its fungi infecting relatives with hopes to identify common viral structural factors necessary for the infection of arthropods. For this purpose, the Hubei.PLV ORF was cloned and then transfected into insect Spodoptera frugiperda (Sf-9) cells using a baculovirus expression system, “bac-to-bac” expression system. The FLAG-tagged capsid proteins were expressed by the Sf-9 cells to be approximately 60 kDa. After ultra-centrifugation in a sucrose gradient, some spontaneous assembly into the expected ~40 nm icosahedral virus-like particles were observed using low resolution scanning electron microscopy. The observed particles were also confirmed by a dynamic light scattering experiment (DLS) and a higher resolution cryo-EM microscope. Thus, the bac-to-bac expression system can be used to produce VLPs from this genus of viruses, and this metagenomically derived virus genome. However, for future success in defining a high-resolution model of this virus, it is recommended that the Sf-9 culture volume is sufficiently high for enough particle production which is necessary for a high-resolution map. The other virus, the Southern pygmy squid Flavilike virus (SpSFV) has been suggested to be the oldest relative of the land based flaviviruses. The SpSFV was found to be the most divergent of the flaviviruses, and to infect invertebrates. Solving for the structure of the SpSFV and comparing it to vertebrate infecting flaviviruses could therefore lead to the identification of factors necessary for the adaptation to vertebrates and thus the humoral immunity by flaviviruses. The soluble E-protein was expressed using the bac-to-bac expression system. The protein was indicated to be multiglycosylated and approximately 50 kDa which is in line with other strains in the genus. Affinity chromatography did not elute this protein, likely due to the His-tag not being spatially available. Cation exchange could elute some protein, but not much from the small ~30 mL culture. To conclude, VLP assembly was confirmed by the Hubei.PLV, thus, solving for the structure is a distinct possibility when a larger Sf-9 culture is used to produce the VLPs. For the SpSFV soluble E-protein, the protein is secreted into the supernatant of the Sf-9 cultures, making purification a possibility. For this, a large Sf-9 culture can be used to produce this protein and then purify it with a cat-ion exchange chromatography.

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  • 16.
    Azinas, Stavros
    et al.
    Molecular recognition and host–pathogen interactions programme, CIC bioGUNE, CIBERehd, Derio, Spain; Biosurfaces Lab, CIC biomaGUNE, San Sebastian, Spain.
    Bano, F.
    Torca, I.
    Bamford, D. H.
    Schwartz, G. A.
    Esnaola, J.
    Oksanen, H. M.
    Richter, R. P.
    Abrescia, N. G.
    Membrane-containing virus particles exhibit the mechanics of a composite material for genome protection2018In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 10, no 16, p. 7769-7779Article in journal (Refereed)
    Abstract [en]

    The protection of the viral genome during extracellular transport is an absolute requirement for virus survival and replication. In addition to the almost universal proteinaceous capsids, certain viruses add a membrane layer that encloses their double-stranded (ds) DNA genome within the protein shell. Using the membrane-containing enterobacterial virus PRD1 as a prototype, and a combination of nanoindentation assays by atomic force microscopy and finite element modelling, we show that PRD1 provides a greater stability against mechanical stress than that achieved by the majority of dsDNA icosahedral viruses that lack a membrane. We propose that the combination of a stiff and brittle proteinaceous shell coupled with a soft and compliant membrane vesicle yields a tough composite nanomaterial well-suited to protect the viral DNA during extracellular transport.

  • 17.
    Badell, Maria Valldeperas
    et al.
    Lund Univ, Phys Chem, Lund, Sweden.;Lund Univ, NanoLund, Lund, Sweden..
    Dabkowska, Aleksandra
    Lund Univ, Phys Chem, Lund, Sweden.;Lund Univ, NanoLund, Lund, Sweden..
    Naidjonoka, Polina
    Lund Univ, Phys Chem, Lund, Sweden..
    Welbourn, Rebecca
    Rutherford Appleton Lab, STFC, ISIS Neutron & Muon Source, Didcot, Oxon, England..
    Pålsson, Gunnar K.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Physics. Inst Laue Langevin, Grenoble, France..
    Barauskas, Justas
    Camurus AB, Lund, Sweden.;Malmo Univ, Biomed Sci, Malmo, Sweden..
    Nylander, Tommy
    Lund Univ, Phys Chem, Lund, Sweden.;Lund Univ, NanoLund, Lund, Sweden..
    Lipid Sponge-Phase Nanoparticles as Carriers for Enzymes2018In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 114, no 3, p. 15A-15AArticle in journal (Other academic)
  • 18. Bailleul, Benjamin
    et al.
    Berne, Nicolas
    Murik, Omer
    Petroutsos, Dimitris
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Physiology and Environmental Toxicology.
    Prihoda, Judit
    Tanaka, Atsuko
    Villanova, Valeria
    Bligny, Richard
    Flori, Serena
    Falconet, Denis
    Krieger-Liszkay, Anja
    Santabarbara, Stefano
    Rappaport, Fabrice
    Joliot, Pierre
    Tirichine, Leila
    Falkowski, Paul G
    Cardol, Pierre
    Bowler, Chris
    Finazzi, Giovanni
    Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms.2015In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 524, no 7565, p. 366-9Article in journal (Refereed)
    Abstract [en]

    Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

  • 19.
    Balzarotti, Francisco
    et al.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Eilers, Yvan
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Gwosch, Klaus C.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Gynnå, Arvid H.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Westphal, Volker
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Stefani, Fernando D.
    Consejo Nacl Invest Cient & Tecn, Ctr Invest Bionanociencias CIBION, Buenos Aires, DF, Argentina.;Univ Buenos Aires, Fac Ciencias Exactas & Nat, Dept Fis, Buenos Aires, DF, Argentina..
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hell, Stefan W.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany.;Max Planck Inst Med Res, Dept Opt Nanoscopy, Heidelberg, Germany.;German Canc Res Ctr, Opt Nanoscopy Div, Heidelberg, Germany..
    Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes2017In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 355, no 6325, p. 606-612Article in journal (Refereed)
    Abstract [en]

    We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolutionmicroscopy, MINFLUXattained similar to 1-nanometer precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.

  • 20. Banijamali, Elnaz
    et al.
    Baronti, Lorenzo
    Becker, Walter
    Sajkowska-Kozielewicz, Joanna J.
    Huang, Ting
    Palka, Christina
    Kosek, David
    Sweetapple, Lara
    Müller, Juliane
    Stone, Michael D.
    Andersson, Emma R.
    Petzold, Katja
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177 Stockholm, Sweden; Stellenbosch Institute for Advanced Study (STIAS), Wallenberg Research Centre at Stellenbosch University, Stellenbosch 7600, South Africa.
    RNA:RNA interaction in ternary complexes resolved by chemical probing2022In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 29, no 3, p. 317-329Article in journal (Refereed)
    Abstract [en]

    RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.

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  • 21.
    Barrozo, Alexandre H.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Carvalho, Alexandra Pires
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Understanding Functional Evolution in the Alkaline Phosphatase Superfamily2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 675A-675AArticle in journal (Other academic)
    Abstract [en]

    Over the past 40 years, it has been demonstrated that many enzymes are capable of promiscuous catalytic activities, facilitating the turnover of more than one chemically distinct substrate. This has been argued to play an important role in enzyme evolution, with highly promiscuous progenitor enzymes evolving under evolutionary pressure to modern day specialists, while still retaining some level of their former promiscuous activities1. This theory has been extensively tested by different experiments using in vitro evolution2. The alkaline phosphatase superfamily members provide a particularly attractive showcase for studying enzyme promiscuity, as they often show reciprocal promiscuity, in that the native reaction for one member is often a side-reaction for another3. While deceptively similar, their catalyzed reactions (cleavage of P-O and S-O bonds) proceed via distinct transition states and protonation requirements4,5. We present detailed computational studies of the promiscuous catalytic activity of three evolutionarily related members: the arylsulfatase from Pseudomonas aeruginosa6, and the phosphonate monoester hydrolases from Burkholderia caryophili7and Rhizobium leguminosarum8. By tracking their structural and electrostatic features, and comparing to other known members of the superfamily, we provide an atomic-level map for functional evolution within this superfamily.

  • 22.
    Bashardanesh, Zahedeh
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Zhang, Haiyang
    University of Science and Technology Beijing, Peoples R China.
    Van der Spoel, David
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Rotational and Translational Diffusion of Proteins as a Function of Concentration2019In: ACS Omega, E-ISSN 2470-1343, Vol. 4, no 24, p. 20654-20664Article in journal (Refereed)
    Abstract [en]

    Atomistic simulations of three different proteins at different concentrations are performed to obtain insight into protein mobility as a function of protein concentration. We report on simulations of proteins from diluted to the physiological water concentration (about 70% of the mass). First, the viscosity was computed and found to increase by a factor of 7-9 going from pure water to the highest protein concentration, in excellent agreement with in vivo nuclear magnetic resonance results. At a physiological concentration of proteins, the translational diffusion is found to be slowed down to about 30% of the in vitro values. The slow-down of diffusion found here using atomistic models is slightly more than that of a hard sphere model that neglects the electrostatic interactions. Interestingly, rotational diffusion of proteins is slowed down somewhat more (by about 80-95% compared to in vitro values) than translational diffusion, in line with experimental findings and consistent with the increased viscosity. The finding that rotation is retarded more than translation is attributed to solvent-separated clustering. No direct interactions between the proteins are found, and the clustering can likely be attributed to dispersion interactions that are stronger between proteins than between protein and water. Based on these simulations, we can also conclude that the internal dynamics of the proteins in our study are affected only marginally under crowding conditions, and the proteins become somewhat more stable at higher concentrations. Simulations were performed using a force field that was tuned for dealing with crowding conditions by strengthening the protein-water interactions. This force field seems to lead to a reproducible partial unfolding of an alpha-helix in one of the proteins, an effect that was not observed in the unmodified force field.

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  • 23.
    Bashardanesh, Zahedeh
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Impact of Dispersion Coefficient on Simulations of Proteins and Organic Liquids2018In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 122, no 33, p. 8018-8027Article in journal (Refereed)
    Abstract [en]

    In the context of studies of proteins under crowding conditions, it was found that there is a tendency of simulated proteins to coagulate in a seemingly unphysical manner. This points to an imbalance in the protein-protein or protein-water interactions. One way to resolve this is to strengthen the protein-water Lennard-Jones interactions. However, it has also been suggested that dispersion interactions may have been systematically overestimated in force fields due to parameterization with a short cutoff. Here, we test this proposition by performing simulations of liquids and of proteins in solution with systematically reduced C-6 (dispersion constant in a 12-6 Lennard-Jones potential) and evaluate the properties. We find that simulations of liquids with either a dispersion correction or explicit long-range Lennard-Jones interactions need little or no correction to the dispersion constant to reproduce the experimental density. For simulations of proteins, a significant reduction in the dispersion constant is needed to reduce the coagulation, however. Because the protein- and liquid force fields share atom types, at least to some extent, another solution for the coagulation problem may be needed, either through including explicit polarization or through strengthening protein-water interactions.

  • 24.
    Bellisario, Alfredo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Deep learning assisted phase retrieval and computational methods in coherent diffractive imaging2024Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In recent years, advances in Artificial Intelligence and experimental techniques have revolutionized the field of structural biology. X-ray crystallography and Cryo-EM have provided unprecedented insights into the structures of biomolecules, while the unexpected success of AlphaFold has opened up new avenues of investigation. However, studying the dynamics of proteins at high resolution remains a significant obstacle, especially for fast dynamics. Single-particle imaging (SPI) or Flash X-ray Imaging (FXI) is an emerging technique that may enable the mapping of the conformational landscape of biological molecules at high resolution and fast time scale. This thesis discusses the potential of SPI/FXI, its challenges, recent experimental successes, and the advancements driving its development. In particular, machine learning and neural networks could play a vital role in fostering data analysis and improving SPI/FXI data processing. In Paper I, we discuss the problem of noise and detector masks in collecting FXI data. I simulated a dataset of diffraction patterns and used it to train a Convolutional Neural Network (U-Net) to restore data by denoising and filling in detector masks. As a natural continuation of this work, I trained another machine learning model in Paper II to estimate 2D protein densities from diffraction intensities. In the final chapter, corresponding to Paper III, we discuss another experimental method, time-resolved Small Angle X-ray Scattering (SAXS), and a new algorithm recently developed for SAXS data, the DENsity from Solution Scattering (DENSS) algorithm. I discuss the potential of DENSS in time-resolved SAXS and its application for structural fitting of AsLOV2, a Light-Oxygen-Voltage (LOV) protein domain from Avena sativa.

    List of papers
    1. Noise reduction and mask removal neural network for X-ray single-particle imaging
    Open this publication in new window or tab >>Noise reduction and mask removal neural network for X-ray single-particle imaging
    2022 (English)In: Journal of applied crystallography, ISSN 0021-8898, E-ISSN 1600-5767, Vol. 55, p. 122-132Article in journal (Refereed) Published
    Abstract [en]

    Free-electron lasers could enable X-ray imaging of single biological macro-molecules and the study of protein dynamics, paving the way for a powerful new imaging tool in structural biology, but a low signal-to-noise ratio and missing regions in the detectors, colloquially termed 'masks', affect data collection and hamper real-time evaluation of experimental data. In this article, the challenges posed by noise and masks are tackled by introducing a neural network pipeline that aims to restore diffraction intensities. For training and testing of the model, a data set of diffraction patterns was simulated from 10 900 different proteins with molecular weights within the range of 10-100 kDa and collected at a photon energy of 8 keV. The method is compared with a simple low-pass filtering algorithm based on autocorrelation constraints. The results show an improvement in the mean-squared error of roughly two orders of magnitude in the presence of masks compared with the noisy data. The algorithm was also tested at increasing mask width, leading to the conclusion that demasking can achieve good results when the mask is smaller than half of the central speckle of the pattern. The results highlight the competitiveness of this model for data processing and the feasibility of restoring diffraction intensities from unknown structures in real time using deep learning methods. Finally, an example is shown of this preprocessing making orientation recovery more reliable, especially for data sets containing very few patterns, using the expansion-maximization-compression algorithm.

    Place, publisher, year, edition, pages
    International Union of Crystallography (IUCr), 2022
    Keywords
    coherent X-ray diffractive imaging (CXDI), free-electron lasers, diffract-then-destroy, protein structures, single particles, XFELs, imaging
    National Category
    Atom and Molecular Physics and Optics
    Identifiers
    urn:nbn:se:uu:diva-467393 (URN)10.1107/S1600576721012371 (DOI)000749998900013 ()35145358 (PubMedID)
    Funder
    Swedish Foundation for Strategic Research , ITM17-0455Swedish Foundation for Strategic Research , 2017-05336Swedish Foundation for Strategic Research , 2018-00234
    Available from: 2022-02-14 Created: 2022-02-14 Last updated: 2024-04-12Bibliographically approved
    2. Deep learning phase retrieval in X-ray single-particle imaging and object support from autocorrelations
    Open this publication in new window or tab >>Deep learning phase retrieval in X-ray single-particle imaging and object support from autocorrelations
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Phase retrieval is an important optimization problem that occurs, for example, in the analysis of coherent diffraction patterns from isolated proteins. All iterative algorithms employed for phase retrieval in this context require some a priori knowledge of the object, usually in the form of a support that describes the extent of the particle. Phase retrieval is a time-consuming task that can often fail, particularly if the support is too loose or of bad quality. In this paper, we present a neural network that can produce low-resolution estimates of the phased object in a fraction of the time that it takes for a full phase retrieval and that can also successfully be used as support for further analysis. Our network is trained on simulated data from biological macromolecules and is thus tailored to the type of data seen in a typical CDI experiment. Other approaches to support finding either require very accurate data without missing regions or require the full phase-retrieval algorithm to be run for a long time. Our network could both speed up off-line analysis, and provide real-time feedback during data collection.

    Keywords
    neural networks, coherent X-ray diffractive imaging (CXDI), phase retrieval algorithm
    National Category
    Atom and Molecular Physics and Optics
    Identifiers
    urn:nbn:se:uu:diva-526434 (URN)
    Available from: 2024-04-10 Created: 2024-04-10 Last updated: 2024-04-12
    3. Microsecond time-resolved X-ray scattering by utilizing MHz repetition rate at second-generation XFELs
    Open this publication in new window or tab >>Microsecond time-resolved X-ray scattering by utilizing MHz repetition rate at second-generation XFELs
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Detecting microsecond structural perturbations in biomolecules has wide relevance inbiology, chemistry, and medicine. Here, we show how MHz repetition rates at X-ray freeelectron lasers (XFELs) can be used to produce microsecond time-series of proteinscattering with exceptionally low noise levels of 0.001%. We demonstrate the approach byderiving new mechanistic insight into Jɑ helix unfolding of a Light-Oxygen-Voltage (LOV)photosensory domain. This time-resolved acquisition strategy is easy to implement andwidely applicable for direct observation of structural dynamics of many biochemicalprocesses. 

    Keywords
    free-electron lasers, time-resolved studies, SAXS, WAXS, sample delivery, XFELs
    National Category
    Biophysics
    Identifiers
    urn:nbn:se:uu:diva-526526 (URN)
    Available from: 2024-04-11 Created: 2024-04-11 Last updated: 2024-04-12
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  • 25.
    Berg, Otto G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Mahmutovic, Anel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    The helical structure of DNA facilitates binding2016In: Journal of Physics A: Mathematical and Theoretical, ISSN 1751-8113, E-ISSN 1751-8121, Vol. 9, no 36, article id 364002Article in journal (Other academic)
    Abstract [en]

    The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.

  • 26.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology.
    Protein Crystallization: Second Edition2009 (ed. 2)Book (Other academic)
  • 27.
    Bergh, Magnus
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Interaction of Ultrashort X-ray Pulses with Material2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Radiation damage limits the resolution in imaging experiments. Damage is caused by energy deposited into the sample during exposure. Ultrashort and extremely bright X-ray pulses from free-electron lasers (FELs) offer the possibility to outrun key damage processes, and temporarily improve radiation tolerance. Theoretical models indicate that high detail-resolutions could be realized on non-crystalline samples with very short pulses, before plasma expansion.

    Studies presented here describe the interaction of a very intense and ultrashort X-ray pulse with material, and investigate boundary conditions for flash diffractive imaging both theoretically and experimentally. In the hard X-ray regime, predictions are based on particle simulations with a continuum formulation that accounts for screening from free electrons.

    First experimental results from the first soft X-ray free-electron laser, the FLASH facility in Hamburg, confirm the principle of flash imaging, and provide the first validation of our theoretical models. Specifically, experiments on nano-fabricated test objects show that an interpretable image can be obtained to high resolution before the sample is vaporized. Radiation intensity in these experiments reached 10^14 W/cm^2, and the temperature of the sample rose to 60000 Kelvin after the 25 femtosecond pulse left the sample. Further experiments with time-delay X-ray holography follow the explosion dynamics over some picoseconds after illumination.

    Finally, this thesis presents results from biological flash-imaging studies on living cells. The model is based on plasma calculations and fluid-like motions of the sample, supported by the time-delay measurements. This study provides an estimate for the achievable resolutions as function of wavelength and pulse length. The technique was demonstrated by our team in an experiment where living cells were exposed to a single shot from the FLASH soft X-ray laser.

    List of papers
    1. Model for the Dynamics of a Water Cluster in an X-ray Free Electron Laser Beam
    Open this publication in new window or tab >>Model for the Dynamics of a Water Cluster in an X-ray Free Electron Laser Beam
    2004 (English)In: Physical Review E. Statistical, Nonlinear, and Soft Matter Physics, ISSN 1539-3755, E-ISSN 1550-2376, Vol. 70, no 5:1, p. 051904-Article in journal (Refereed) Published
    Abstract [en]

    A microscopic sample placed into a focused x-ray free electron laser beam will explode due to strong ionization on a femtosecond time scale. The dynamics of this Coulomb explosion has been modeled by Neutze et al. [Nature (London) 406, 752 (2000)] for a protein, using computer simulations. The results suggest that by using ultrashort exposures, structural information may be collected before the sample is destroyed due to radiation damage. In this paper a method is presented to include the effect of screening by free electrons in the sample in a molecular dynamics simulation. The electrons are approximated by a classical gas, and the electron distribution is calculated iteratively from the Poisson-Boltzmann equation. Test simulations of water clusters reveal the details of the explosion dynamics, as well as the evolution of the free electron gas during the beam exposure. We find that inclusion of the electron gas in the model slows down the Coulomb explosion. The hydrogen atoms leave the sample faster than the oxygen atoms, leading to a double layer of positive ions. A considerable electron density is located between these two layers. The fact that the hydrogens are found to explode much faster than the oxygens means that the diffracting part of the sample stays intact somewhat longer than the sample as a whole.

    Keywords
    Computer Simulation, Electrons, Lasers, Models
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-96323 (URN)15600653 (PubMedID)
    Available from: 2007-10-24 Created: 2007-10-24 Last updated: 2017-12-14Bibliographically approved
    2. Soft-x-ray free-electron-laser interaction with materials
    Open this publication in new window or tab >>Soft-x-ray free-electron-laser interaction with materials
    2007 (English)In: Physical Review E. Statistical, Nonlinear, and Soft Matter Physics: Statistical Physics, Plasmas, Fluids, and Related Interdisciplinary Topics, ISSN 1063-651X, E-ISSN 1095-3787, Vol. 76, no 4, p. 046403-Article in journal (Refereed) Published
    Abstract [en]

    Soft-x-ray free-electron lasers have enabled materials studies in which structural information is obtained faster than the relevant probe-induced damage mechanisms. We present a continuum model to describe the damage process based on hot-dense plasma theory, which includes a description of the energy deposition in the samples, the subsequent dynamics of the sample, and the detector signal. We compared the model predictions with experimental data and mostly found reasonable agreement. In view of future free-electron-laser performance, the model was also used to predict damage dynamics of samples and optical elements at shorter wavelengths and larger photon fluences than currently available.

    National Category
    Physical Sciences
    Identifiers
    urn:nbn:se:uu:diva-96324 (URN)10.1103/PhysRevE.76.046403 (DOI)000250622100073 ()
    Available from: 2007-10-24 Created: 2007-10-24 Last updated: 2017-12-14Bibliographically approved
    3. Force Field Benchmark of Organic Liquids: Density, Enthalpy of Vaporization, Heat Capacities, Surface Tension, Isothermal Compressibility, Volumetric Expansion Coefficient, and Dielectric Constant
    Open this publication in new window or tab >>Force Field Benchmark of Organic Liquids: Density, Enthalpy of Vaporization, Heat Capacities, Surface Tension, Isothermal Compressibility, Volumetric Expansion Coefficient, and Dielectric Constant
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    2007 (English)In: Physical Review Letters, ISSN 0031-9007, E-ISSN 1079-7114, Vol. 98, no 14, p. 145502-Article in journal (Refereed) Published
    Abstract [en]

    At the recently built FLASH x-ray free-electron laser, we studied the reflectivity of Si/C multilayers with fluxes up to 3×1014W/cm2. Even though the nanostructures were ultimately completely destroyed, we found that they maintained their integrity and reflectance characteristics during the 25-fs-long pulse, with no evidence for any structural changes over lengths greater than 3Å. This experiment demonstrates that with intense ultrafast pulses, structural damage does not occur during the pulse, giving credence to the concept of diffraction imaging of single macromolecules.

    Keywords
    X-ray effects, Optical properties of low-dimensional, mesoscopic, and nanoscale materials and structures
    National Category
    Biological Sciences Physical Sciences
    Identifiers
    urn:nbn:se:uu:diva-96325 (URN)10.1103/PhysRevLett.98.145502 (DOI)000245512100037 ()
    Note


    Available from: 2007-10-24 Created: 2007-10-24 Last updated: 2017-12-14Bibliographically approved
    4. Feasibility of imaging living cells at subnanometer resolutions by ultrafast X-ray diffraction
    Open this publication in new window or tab >>Feasibility of imaging living cells at subnanometer resolutions by ultrafast X-ray diffraction
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    2008 (English)In: Quarterly reviews of biophysics (Print), ISSN 0033-5835, E-ISSN 1469-8994, Vol. 41, no 3-4, p. 181-204Article, review/survey (Refereed) Published
    Abstract [en]

    Detailed structural investigations on living cells are problematic because existing structural methods cannot reach high resolutions on non-reproducible objects. Illumination with an ultrashort and extremely bright X-ray pulse can outrun key damage processes over a very short period. This can be exploited to extend the diffraction signal to the highest possible resolution in flash diffraction experiments. Here we present an analysis or the interaction of a very intense and very short X-ray pulse with a living cell, using a non-equilibrium population kinetics plasma code with radiation transfer. Each element in the evolving plasma is modeled by numerous states to monitor changes in the atomic populations as a function of pulse length, wavelength, and fluence. The model treats photoionization, impact ionization, Auger decay, recombination, and inverse bremsstrahlung by solving rate equations in a self-consistent manner and describes hydrodynamic expansion through the ion sound speed, The results show that subnanometer resolutions could be reached on micron-sized cells in a diffraction-limited geometry at wavelengths between 0.75 and 1.5 nm and at fluences of 10(11)-10(12) photonS mu M (2) in less than 10 fs. Subnanometer resolutions could also be achieved with harder X-rays at higher fluences. We discuss experimental and computational strategies to obtain depth information about the object in flash diffraction experiments.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-96326 (URN)10.1017/S003358350800471X (DOI)000262098500001 ()
    Available from: 2007-10-24 Created: 2007-10-24 Last updated: 2022-01-28Bibliographically approved
    5. Interaction of Ultrashort X-ray Pulses with B4C, SiC and Si
    Open this publication in new window or tab >>Interaction of Ultrashort X-ray Pulses with B4C, SiC and Si
    2008 (English)In: Physical Review E. Statistical, Nonlinear, and Soft Matter Physics: Statistical Physics, Plasmas, Fluids, and Related Interdisciplinary Topics, ISSN 1063-651X, E-ISSN 1095-3787, Vol. 77, no 2, p. 026404-1-026404-8Article in journal (Refereed) Published
    Abstract [en]

    The interaction of 32.5 and 6 nm ultrashort x-ray pulses with the solid materials B4C, SiC, and Si is simulated with a nonlocal thermodynamic equilibrium radiation transfer code. We study the ionization dynamics as a function of depth in the material and modifications of the opacity during irradiation, and estimate the crater depth. Furthermore, we compare the estimated crater depth with experimental data, for fluences up to 2.2 J/cm(2). Our results show that, at 32.5 nm irradiation, the opacity changes by less than a factor of 2 for B4C and Si and by a factor of 3 for SiC, for fluences up to 200 J/cm(2). At a laser wavelength of 6 nm, the model predicts a dramatic decrease in opacity due to the weak inverse bremsstrahlung, increasing the crater depth for high fluences.

    Keywords
    boron compounds, bremsstrahlung, elemental semiconductors, high-speed optical techniques, ionisation, laser beam effects, opacity, silicon, silicon compounds, thermodynamics, wide band gap semiconductors, X-ray effects
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-96327 (URN)10.1103/PhysRevE.77.026404 (DOI)000253763800053 ()18352130 (PubMedID)
    Available from: 2007-10-24 Created: 2007-10-24 Last updated: 2022-01-28Bibliographically approved
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  • 28.
    Berntsson, Elina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    The effect of Edaravone on Amyloid beta aggregation2019Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    Alzheimer’s disease (AD) is a devastating neurodegenerative disease that affect millions of people worldwide. Aggregation of Amyloid-β (Aβ) monomers create toxic oligomers that can interact with cellular membranes and disturb cellular functions, resulting in cell death and neurological dysfunction. Increased levels of oxidative stress have been shown in the brains of AD patients, something that besides the obvious cell and tissue toxicity, also favors the amyloidogenic pathway and generates more Aβ monomers. Here we show that Edaravone, a free radical scavenger can affect the aggregation rate of different lengths of Aβ. We show that Aβ-40 that is more commonly found in vivo aggregates faster with addition of Edaravone, while Aβ-42 aggregates slower or not at all. These findings add up to previous findings where free radical scavengers and antioxidants such as Edaravone have been suggested as a potential treatment in Alzheimer’s disease.

  • 29.
    Bielecki, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hantke, Max F.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Daurer, Benedikt J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Reddy, Hemanth K. N.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hasse, Dirk
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Larsson, Daniel S. D.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Gunn, Laura H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Svenda, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Munke, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Sellberg, Jonas A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Flueckiger, Leonie
    Pietrini, Alberto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Nettelblad, Carl
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Lundholm, Ida
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Carlsson, Gunilla
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Okamoto, Kenta
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Molecular and Condensed Matter Physics.
    Westphal, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Kulyk, Olena
    Higashiura, Akifumi
    van der Schot, Gijs
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Loh, Ne-Te Duane
    Wysong, Taylor E.
    Bostedt, Christoph
    Gorkhover, Tais
    Iwan, Bianca
    Seibert, M. Marvin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Osipov, Timur
    Walter, Peter
    Hart, Philip
    Bucher, Maximilian
    Ulmer, Anatoli
    Ray, Dipanwita
    Carini, Gabriella
    Ferguson, Ken R.
    Andersson, Inger
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Andreasson, Jakob
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Maia, Filipe R. N. C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Electrospray sample injection for single-particle imaging with x-ray lasers2019In: Science Advances, E-ISSN 2375-2548, Vol. 5, no 5, article id eaav8801Article in journal (Refereed)
  • 30.
    Binti Shamsudin, Yasmin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Department of Chemistry, Stanford University, Stanford, CA, 94305, USA.
    Walker, Alice R.
    Jones, Chey M.
    Martínez, Todd J.
    Boxer, Steven G.
    Simulation-guided engineering of split GFPs with efficient β-strand photodissociation2023In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1, article id 7401Article in journal (Refereed)
    Abstract [en]

    Green fluorescent proteins (GFPs) are ubiquitous for protein tagging and live-cell imaging. Split-GFPs are widely used to study protein-protein interactions by fusing proteins of interest to split GFP fragments that create a fluorophore upon typically irreversible complementation. Thus, controlled dissociation of the fragments is desirable. Although we have found that split strands can be photodissociated, the quantum efficiency of light-induced photodissociation of split GFPs is low. Traditional protein engineering approaches to increase efficiency, including extensive mutagenesis and screening, have proved difficult to implement. To reduce the search space, key states in the dissociation process are modeled by combining classical and enhanced sampling molecular dynamics with QM/MM calculations, enabling the rational design and engineering of split GFPs with up to 20-fold faster photodissociation rates using non-intuitive amino acid changes. This demonstrates the feasibility of modeling complex molecular processes using state-of-the-art computational methods, and the potential of integrating computational methods to increase the success rate in protein engineering projects.

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  • 31.
    Blokzijl, Andries
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Nong, Rachel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Darmanis, S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Hertz, E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Protein biomarker validation via proximity ligation assays2014In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1844, no 5, p. 933-939Article, review/survey (Refereed)
    Abstract [en]

    The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PIA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. (C) 2013 Elsevier B.V. All rights reserved.

  • 32.
    Bortot, Leandro Oliveira
    et al.
    Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Phys Biol Lab, Ave Cafe S-N, BR-14040903 Ribeirao Preto, SP, Brazil.
    Bashardanesh, Zahedeh
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Making Soup: Preparing and Validating Models of the Bacterial Cytoplasm for Molecular Simulation2020In: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 60, no 1, p. 322-331Article in journal (Refereed)
    Abstract [en]

    Biomolecular crowding affects the biophysical and biochemical behavior of macromolecules compared with the dilute environment in experiments on isolated proteins. Computational modeling and simulation are useful tools to study how crowding affects the structural dynamics and biological properties of macromolecules. With increases in computational power, modeling and simulation of large-scale all-atom explicit-solvent models of the prokaryote cytoplasm have now become possible. In this work, we built an atomistic model of the cytoplasm of Escherichia coli composed of 1.5 million atoms and submitted it to a total of 3 mu s of molecular dynamics simulations. The model consisted of eight different proteins representing about 50% of the cytoplasmic proteins and one type of t-RNA molecule. Properties of biomolecules under crowding conditions were compared with those from simulations of the individual compounds under dilute conditions. The simulation model was found to be consistent with experimental data about the diffusion coefficient and stability of macromolecules under crowded conditions. In order to stimulate further work, we provide a Python script and a set of files to enable other researchers to build their own E. coli cytoplasm models to address questions related to crowding.

  • 33.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Larsson, Jimmy
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Antibiotic perseverance increases the risk of resistance development2023In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 120, no 2, article id e2216216120Article in journal (Refereed)
    Abstract [en]

    The rise of antibiotic-resistant bacterial infections poses a global threat. Antibiotic resistance development is generally studied in batch cultures which conceals the heterogeneity in cellular responses. Using single-cell imaging, we studied the growth response of Escherichia coli to sub-inhibitory and inhibitory concentrations of nine antibiotics. We found that the heterogeneity in growth increases more than what is expected from growth rate reduction for three out of the nine antibiotics tested. For two antibiotics (rifampicin and nitrofurantoin), we found that sub-populations were able to maintain growth at lethal antibiotic concentrations for up to 10 generations. This perseverance of growth increased the population size and led to an up to 40-fold increase in the frequency of antibiotic resistance mutations in gram-negative and gram-positive species. We conclude that antibiotic perseverance is a common phenomenon that has the potential to impact antibiotic resistance development across pathogenic bacteria.

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  • 34.
    Cai, Yixiao
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Lendel, Christofer
    Österlund, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.
    Kasrayan, Alex
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Nikolajeff, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Karlsson, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Bergström, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Changes in secondary structure of α-synuclein during oligomerization induced by reactive aldehydes.2015In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 464, no 1, p. 336-341Article in journal (Refereed)
    Abstract [en]

    The oxidative stress-related reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) have been shown to promote formation of α-synuclein oligomers in vitro. However, the changes in secondary structure of α-synuclein and the kinetics of the oligomerization process are not known and were the focus of this study. Size exclusion chromatography showed that after 1 h of incubation, HNE induced the formation of an oligomeric α-synuclein peak with a molecular weight of about ∼2000 kDa, which coincided with a decreasing ∼50 kDa monomeric peak. With prolonged incubation (up to 24 h) the oligomeric peak became the dominating molecular species. In contrast, in the presence of ONE, a ∼2000 oligomeric peak was exclusively observed after 15 min of incubation and this peak remained constant with prolonged incubation. Western blot analysis of HNE-induced α-synuclein oligomers showed the presence of monomers (15 kDa), SDS-resistant low molecular (30-160 kDa) and high molecular weight oligomers (≥260 kDa), indicating that the oligomers consisted of both covalent and non-covalent protein. In contrast, ONE-induced α-synuclein oligomers only migrated as covalent cross-linked high molecular-weight material (≥300 kDa). Both circular dichroism (CD) and Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy showed that the formation of HNE- and ONE-induced oligomers coincided with a spectral change from random coil to β-sheet. However, ONE-induced α-synuclein oligomers exhibited a slightly higher degree of β-sheet. Taken together, our results indicate that both HNE and ONE induce a change from random coil to β-sheet structure that coincides with the formation of α-synuclein oligomers. Albeit through different kinetic pathways depending on the degree of cross-linking.

  • 35.
    Caleman, Carl
    et al.
    Physik Department E17, Technische Universität München.
    Ortiz, Carlos
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Materials Science, Materials Theory.
    Marklund, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Bultmark, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Materials Science, Materials Theory.
    Gabrysch, Markus
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Electricity.
    Parak, F. G.
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Klintenberg, Mattias
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Materials Science, Materials Theory.
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Radiation damage in biological material: electronic properties and electron impact ionization in urea2009In: Europhysics letters, ISSN 0295-5075, E-ISSN 1286-4854, Vol. 85, no 1, p. 18005-Article in journal (Refereed)
    Abstract [en]

    Radiation damage is an unavoidable process when performing structural investigations of biological macromolecules with X-rays. In crystallography this process can be limited through damage distribution in a crystal, while for single molecular imaging it can be outrun by employing short intense pulses. Secondary electron generation is crucial during damage formation and we present a study of urea, as model for biomaterial. From first principles we calculate the band structure and energy loss function, and subsequently the inelastic electron cross-section in urea. Using Molecular Dynamics simulations, we quantify the damage and study the magnitude and spatial extent of the electron cloud coming from an incident electron, as well as the dependence with initial energy.

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  • 36.
    Cardoch, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Computational study of single protein sensing using nanopores2020Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Identifying the protein content in a cell in a fast and reliable manner has become a relevant goal in the field of proteomics. This thesis computationally explores the potential for silicon nitride nanopores to sense and distinguish single miniproteins, which are small domains that promise to facilitate the systematic study of larger proteins. Sensing and identification of these biomolecules using nanopores happens by studying modulations in ionic current during translocation. The approach taken in this work was to study two miniproteins of similar geometry, using a cylindrical-shaped pore. I employed molecular mechanics to compare occupied pore currents computed based on the trajectory of ions. I further used density functional theory along with relative surface accessibility values to compute changes in interaction energies for single amino acids and obtain relative dwell times. While the protein remained inside the nanopore, I found no noticeable differences in the occupied pore currents of the two miniproteins for systems subject to 0.5 and 1.0 V bias voltages. Dwell times were estimated based on the translocation time of a protein that exhibits no interaction with the pore walls. I found that both miniproteins feel an attractive force to the pore wall and estimated their relative dwell times to differ by one order of magnitude. This means even in cases where two miniproteins are indistinguishable by magnitude changes in the ionic current, the dwell time might still be used to identify them. This work was an initial investigation that can be further developed to increase the accuracy of the results and be expanded to assess other miniproteins with the goal to aid future experimental work.

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  • 37.
    Carlsson, Gunilla
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Crystallography in Four Dimensions: Methods and Applications2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The four-electron reduction of dioxygen to water is the most exothermic non-photochemical reaction available to biology. A detailed molecular description of this reaction is needed to understand oxygen-based redox processes. Horseradish peroxidase (HRP) is a haem-containing redox enzyme capable of catalysing the reduction of dioxygen to water. We developed instrumentation and experimental methodology to capture and characterise by X-ray crystallography transient reaction intermediates in this reaction.

    An instrument was designed (“the vapour stream system”) to facilitate reaction initiation, monitoring and intermediate trapping. In combination with single crystal microspectrophotometry, it was used to obtain conditions for capturing a reactive dioxygen complex in HRP. X-ray studies on oxidised intermediates can be difficult for various reasons. Electrons re-distributed in the sample through the photoelectric effect during X-ray exposure can react with high-valency intermediates. In order to control such side reactions during data collection, we developed a new method based on an angle-resolved spreading of the X-ray dose over many identical crystals. Composite data sets built up from small chunks of data represent crystal structures which received different X-ray doses. As the number of electrons liberated in the crystal is dose dependent, this method allows us to observe and drive redox reactions electron-by-electron in the crystal, using X-rays.

    The methods developed here were used to obtain a three-dimensional movie on the X-ray-driven reduction of dioxygen to water in HRP. Separate experiments established high resolution crystal structures for all intermediates, showing such structures with confirmed redox states for the first time.

    Activity of HRP is influenced by small molecule ligands, and we also determined the structures of HRP in complex with formate, acetate and carbon monoxide.

    Other studies established conditions for successfully trapping the M-intermediate in crystals of mutant bacteriorhodopsin, but the poor diffraction quality of these crystals prevented high-resolution structural studies.

    List of papers
    1. Protein crystallography in a vapour steam: data collection, reaction initiation and intermediate trapping in naked hydrated protein crystals
    Open this publication in new window or tab >>Protein crystallography in a vapour steam: data collection, reaction initiation and intermediate trapping in naked hydrated protein crystals
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    2002 (English)In: Journal of applied crystallography, ISSN 0021-8898, E-ISSN 1600-5767, Vol. 35, no 1, p. 113-116Article in journal (Refereed) Published
    Abstract [en]

    A procedure is presented for experiments on naked unfrozen protein crystals with the crystal mounted in a conventional cryo-loop and surrounded by a stream of a wet gas. The composition and temperature of the vapour stream can be adjusted to keep the crystal without deterioration for many hours. The arrangement allows (i) for rapidly testing crystals for diffraction before freezing, (ii) for data collection between 268-303 K with greatly reduced background, (iii) for the controlled drying or wetting of crystals, (iv) for the anaerobic manipulation of protein crystals, and (v) for the introduction of gaseous or volatile ingredients and reactants into the crystal. The technique offers new experimental possibilities, e.g. in time-resolved structural studies. Reaction initiation in many protein crystals can be achieved by changing the composition of the vapour stream to create a new chemical environment around the crystal and to introduce substrates/reactants either in the gas phase or as microdroplets. Spectral changes during such reactions can be monitored by single-crystal microspectrophotometry, and, once an intermediate has been detected at high concentrations, the crystal can be frozen, e.g. by rapidly switching the warm vapour stream to a cryogenically cooled helium or nitrogen jet. Representative examples are presented in this paper.

    National Category
    Medical and Health Sciences
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-91905 (URN)10.1107/S0021889801020702 (DOI)
    Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2017-12-14Bibliographically approved
    2. Defining redox state of X-ray crystal structures by single-crystal ultraviolet visible microspectrophotometry
    Open this publication in new window or tab >>Defining redox state of X-ray crystal structures by single-crystal ultraviolet visible microspectrophotometry
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    2002 (English)In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 353, p. 301-318Article in journal (Refereed) Published
    Abstract [en]

    Exciting results have been emerging from the field of single-crystal X-ray crystallography, giving unprecedented detail of freeze-trapped reaction intermediates from important classes of macromolecules that contain chromophores. These structures have been coupled with single-crystal UV-visible microspectrophotometry. This has defined the distinct catalytic intermediates present in the crystal structures, allowing the correlation of electronic transitions with the observed structural transitions. Of particular note is that many of these structures have been generated “on the fly” during kinetic turnover in the crystal. Most enzymatic reactions proceed through distinct catalytic intermediates that, under favorable conditions, may accumulate transiently in the crystal during turnover. In some cases, the physical constraints of the contacts within crystals may also lead to a significant slowing of the reaction at certain points along the pathway where conformational changes are required. This can lead to a transient build-up of spectrally distinct intermediates in the crystal that can be trapped by flash freezing in liquid nitrogen, allowing a complete single-crystal data set to be collected to the highest possible resolution at a later time. Similar build-up of intermediates may be achieved by altering the pH, temperature, or the solvent environment around the protein in the crystal, or by producing engineered variants that build up an intermediate of interest. The chapter focuses on the technical considerations required to carry out UV-visible microspectroscopy of single crystals.

    National Category
    Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-91906 (URN)10.1016/S0076-6879(02)53057-3 (DOI)000176466500027 ()
    Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2017-12-14Bibliographically approved
    3. The catalytic pathway of horseradish peroxidase at high resolution
    Open this publication in new window or tab >>The catalytic pathway of horseradish peroxidase at high resolution
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    2002 In: Nature, Vol. 417, p. 463-468Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-91907 (URN)
    Available from: 2004-09-01 Created: 2004-09-01Bibliographically approved
    4. Complexes of horseradish peroxidase with formate, acetate and carbon monoxide
    Open this publication in new window or tab >>Complexes of horseradish peroxidase with formate, acetate and carbon monoxide
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    2005 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 2, p. 635-642Article in journal (Refereed) Published
    Abstract [en]

    Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the “hydrated” form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP−acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463−468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron−ligand distances of 2.3 and 1.8 Å, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 Å from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-91908 (URN)10.1021/bi0483211 (DOI)
    Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2017-12-14Bibliographically approved
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  • 38.
    Carter, Sarah-Sophia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Atif, Abdul Raouf
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Diez-Escudero, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Grape, Maja
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ginebra, Maria-Pau
    Biomaterials, Biomechanics and Tissue Engineering Group, Departament de Ciència i Enginyeria de Materials, Universitat Politècnica de Catalunya (UPC), 08930, Barcelona, Spain.;Barcelona Research Center in Multiscale Science and Engineering, Universitat Politècnica de Catalunya, 08930, Barcelona, Spain.;Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10-12, 08028, Barcelona, Spain..
    Tenje, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mestres, Gemma
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering. Uppsala University, Science for Life Laboratory, SciLifeLab.
    A microfluidic-based approach to investigate the inflammatory response of macrophages to pristine and drug-loaded nanostructured hydroxyapatite2022In: Materials Today Bio, ISSN 2590-0064, Vol. 16, article id 100351Article in journal (Refereed)
    Abstract [en]

    The in vitro biological characterization of biomaterials is largely based on static cell cultures. However, for highly reactive biomaterials such as calcium-deficient hydroxyapatite (CDHA), this static environment has limitations. Drastic alterations in the ionic composition of the cell culture medium can negatively affect cell behavior, which can lead to misleading results or data that is difficult to interpret. This challenge could be addressed by a microfluidics-based approach (i.e. on-chip), which offers the opportunity to provide a continuous flow of cell culture medium and a potentially more physiologically relevant microenvironment. The aim of this work was to explore microfluidic technology for its potential to characterize CDHA, particularly in the context of inflammation. Two different CDHA substrates (chemically identical, but varying in microstructure) were integrated on-chip and subsequently evaluated. We demonstrated that the on-chip environment can avoid drastic ionic alterations and increase protein sorption, which was reflected in cell studies with RAW 264.7 macrophages. The cells grown on-chip showed a high cell viability and enhanced proliferation compared to cells maintained under static conditions. Whereas no clear differences in the secretion of tumor necrosis factor alpha (TNF-α) were found, variations in cell morphology suggested a more anti-inflammatory environment on-chip. In the second part of this study, the CDHA substrates were loaded with the drug Trolox. We showed that it is possible to characterize drug release on-chip and moreover demonstrated that Trolox affects the TNF-α secretion and morphology of RAW 264.7 ​cells. Overall, these results highlight the potential of microfluidics to evaluate (bioactive) biomaterials, both in pristine form and when drug-loaded. This is of particular interest for the latter case, as it allows the biological characterization and assessment of drug release to take place under the same dynamic in vitro environment.

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  • 39.
    Cavallaro, Sara
    et al.
    KTH Royal Inst Technol, Dept Appl Phys, Sch Engn Sci, S-10691 Stockholm, Sweden.
    Pevere, Federico
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Electrical Engineering, Solid-State Electronics.
    Stridfeldt, Fredrik
    KTH Royal Inst Technol, Dept Appl Phys, Sch Engn Sci, S-10691 Stockholm, Sweden.
    Görgens, André
    Karolinska Inst, Dept Lab Med, Clin Res Ctr, S-17177 Stockholm, Sweden; Evox Therapeut Ltd, Oxford Sci Pk, Oxford OX4 4HG, England; Univ Duisburg Essen, Univ Hosp Essen, Inst Transfus Med, D-45141 Essen, Germany.
    Paba, Carolina
    Politecn Torino, I-10129 Turin, Italy.
    Sahu, Siddharth S.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Electrical Engineering, Solid-State Electronics.
    Mamand, Doste R.
    Karolinska Inst, Dept Lab Med, Clin Res Ctr, S-17177 Stockholm, Sweden.
    Gupta, Dhanu
    Karolinska Inst, Dept Lab Med, Clin Res Ctr, S-17177 Stockholm, Sweden; Evox Therapeut Ltd, Oxford Sci Pk, Oxford OX4 4HG, England.
    El Andaloussi, Samir
    Karolinska Inst, Dept Lab Med, Clin Res Ctr, S-17177 Stockholm, Sweden; Evox Therapeut Ltd, Oxford Sci Pk, Oxford OX4 4HG, England.
    Linnros, Jan
    KTH Royal Inst Technol, Dept Appl Phys, Sch Engn Sci, S-10691 Stockholm, Sweden.
    Dev, Apurba
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Electrical Engineering, Solid-State Electronics. KTH Royal Inst Technol, Dept Appl Phys, Sch Engn Sci, S-10691 Stockholm, Sweden.
    Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features2021In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 17, no 14, article id 2008155Article in journal (Refereed)
    Abstract [en]

    Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.

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  • 40.
    Cederfelt, Daniela
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Structural studies of drug targets and a drug metabolizing enzyme2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The work presented in this thesis describes how structural information about a protein can be acquired, and how it can be used to answer scientific questions about proteins’ function, their dynamic behaviour and their interactions with other proteins or ligands.

    The catalytic function of the pyrimidine-degrading, drug metabolizing enzyme β-ureidopropionase (βUP) is dependent on the shift between oligomeric states. Substitution of amino acids H173 and H307 in the dimer-dimer interface and E207Q in the active site revealed that these are crucial for βUP activation. Inhibition studies of substrate-and product analogues allowed for a hypothesis that the ability to interact with F205 might distinguish activators from inhibitors. The first structure of the activated higher oligomer state of human βUP was determined using cryogenic electron microscopy, and confirmed that the closed entrance loop conformations and dimer-dimer interfaces are conserved between HsβUP and DmβUP. 

    Interactions between the epigenetic drug target SET and MYND domain containing protein 3 (SMYD3) and possible inhibitors were investigated. A crystal structure confirmed the covalent bond of a rationally designed, targeted inhibitor to C186 in the active site of SMYD3. A new allosteric binding site was discovered using a biosensor screen with a blocked active site. Crystal structures revealed the location of the new binding site, and the binding mode of the (S)-and (R) enantiomers of the allosteric inhibitor. Lastly, a fragment based drug discovery approach was taken, co-crystallizing and soaking SMYD3 with hits from a fragment screen. This resulted in four crystal structures with weak electron density of fragments at several locations in the enzyme. 

    The dynamic acetylcholine binding protein (AChBP) is a homologue of a Cys-loop type ligand gated ion channel. Hits from various biosensor screens, of which some indicated conformational changes, were co-crystallized with AChBP. Seven crystal structures of AChBP in complex with hit compounds from the biophysical screens were determined. Small conformational changes in the Cys-loop were detected in several of the crystal structures, coinciding with the results from the biosensor screens.

    In these studies, we explore new strategies for the investigation of the function and regulation of proteins relevant in drug discovery and optimization.

    List of papers
    1. The Allosteric Regulation of Β-Ureidopropionase Depends on Fine-Tuned Stability of Active-Site Loops and Subunit Interfaces
    Open this publication in new window or tab >>The Allosteric Regulation of Β-Ureidopropionase Depends on Fine-Tuned Stability of Active-Site Loops and Subunit Interfaces
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    2023 (English)In: Biomolecules, E-ISSN 2218-273X, Vol. 13, no 12, article id 1763Article in journal (Refereed) Published
    Abstract [en]

    The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human β-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer-dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer-dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human β-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer-dimer interfaces are highly conserved between human and fruit fly enzymes.

    Place, publisher, year, edition, pages
    MDPI, 2023
    Keywords
    5-fluorouracil metabolism, allosteric regulation, amidohydrolase, cryo-electron microscopy, pyrimidine degradation
    National Category
    Structural Biology Biochemistry and Molecular Biology
    Research subject
    Biochemistry; Biology with specialization in Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-508072 (URN)10.3390/biom13121763 (DOI)001130877800001 ()38136634 (PubMedID)
    Funder
    Carl Tryggers foundation , CTS18:84
    Note

    Authors in the list of papers of Daniela Cederfelt's thesis: Daniela Cederfelt, Dilip Badjugar, Ayan Musse, Dirk Maurer, Berhard Lohkamp, Doreen Dobritzsh

    Title in the list of papers of Daniela Cederfelt's thesis: The allosteric regulation of the anticancer drug-metabolizing β-ureidopropionase depends on fine-tuned active-site loop andsubunit interface stability

    Available from: 2023-07-19 Created: 2023-07-19 Last updated: 2024-02-16Bibliographically approved
    2. Discovery of the 4-aminopiperidine-based compound EM127 for the site-specific covalent inhibition of SMYD3
    Open this publication in new window or tab >>Discovery of the 4-aminopiperidine-based compound EM127 for the site-specific covalent inhibition of SMYD3
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    2022 (English)In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 243, article id 114683Article in journal (Refereed) Published
    Abstract [en]

    Recent findings support the hypothesis that inhibition of SMYD3 methyltransferase may be a therapeutic avenue for some of the deadliest cancer types. Herein, active site-selective covalent SMYD3 inhibitors were designed by introducing an appropriate reactive cysteine trap into reversible first-generation SMYD3 inhibitors. The 4-amino-piperidine derivative EM127 (11C) bearing a 2-chloroethanoyl group as reactive warhead showed selectivity for Cys186, located in the substrate/histone binding pocket. Selectivity towards Cys186 was retained even at high inhibitor/enzyme ratio, as shown by mass spectrometry. The mode of interaction with the SMYD3 substrate/ histone binding pocket was revealed by crystallographic studies. In enzymatic assays, 11C showed a stronger SMYD3 inhibitory effect compared to the reference inhibitor EPZ031686. Remarkably, 11C attenuated the proliferation of MDA-MB-231 breast cancer cell line at the same low micromolar range of concentrations that reduced SMYD3 mediated ERK signaling in HCT116 colorectal cancer and MDA-MB-231 breast cancer cells. Furthermore, 11C (5 mu M) strongly decreased the steady-state mRNA levels of genes important for tumor biology such as cyclin dependent kinase 2, c-MET, N-cadherin and fibronectin 1, all known to be regulated, at least in part, by SMYD3. Thus, 11C is as a first example of second generation SMYD3 inhibitors; this agent represents a covalent and a site specific SMYD3 binder capable of potent and prolonged attenuation of methyltransferase activity.

    Place, publisher, year, edition, pages
    Elsevier, 2022
    Keywords
    Covalent inhibitor, SMYD3, Lysine methyltransferase, Epigenetic inhibitors, Cancer target therapy
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-487227 (URN)10.1016/j.ejmech.2022.114683 (DOI)000862667200009 ()36116234 (PubMedID)
    Funder
    EU, Horizon 2020
    Available from: 2022-11-14 Created: 2022-11-14 Last updated: 2023-08-08Bibliographically approved
    3. Discovery of an allosteric ligand binding site in SMYD3 lysine methyltransferase
    Open this publication in new window or tab >>Discovery of an allosteric ligand binding site in SMYD3 lysine methyltransferase
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    2021 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 22, no 9, p. 1597-1608Article in journal (Refereed) Published
    Abstract [en]

    SMYD3 is a multifunctional epigenetic enzyme with lysine methyl transferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened using a biosensor-based competition assay. Diperodon was identified as an allosteric ligand. The ( R )-and ( S )-enantiomers of the racemic drug were isolated and their affinities determined ( K D > = 42 and 84 ÎŒM). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although HSP90-SMYD3 binding was confirmed ( K D = 13 ÎŒM). The allosteric site appears to be druggable and suitable for exploration of non-catalytic SMYD3 functions and therapeutics with new mechanisms of action.

    Keywords
    SMYD3, Lysine methyl transferase, Surface plasmon resonance, screening, diperodon
    National Category
    Biophysics
    Identifiers
    urn:nbn:se:uu:diva-429944 (URN)10.1002/cbic.202000736 (DOI)000616767800001 ()33400854 (PubMedID)
    Available from: 2021-01-14 Created: 2021-01-14 Last updated: 2023-10-31Bibliographically approved
    4. Introduction to kinetic screening for the identification of weak fragment hits – mapping ligand binding sites in SMYD3
    Open this publication in new window or tab >>Introduction to kinetic screening for the identification of weak fragment hits – mapping ligand binding sites in SMYD3
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    FBDD, Drug discovery, SAR, Screening, Epigenetics, SPR, Compound optimization.
    National Category
    Biophysics
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-508761 (URN)
    Available from: 2023-08-08 Created: 2023-08-08 Last updated: 2023-08-08
    5. Discovery of fragments inducing conformational effects in dynamic proteins using a second-harmonic generation biosensor
    Open this publication in new window or tab >>Discovery of fragments inducing conformational effects in dynamic proteins using a second-harmonic generation biosensor
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    2021 (English)In: RSC Advances, E-ISSN 2046-2069, Vol. 11, no 13, p. 7527-7537Article in journal (Refereed) Published
    Abstract [en]

    Biophysical screening of compound libraries for the identification of ligands that interact with a protein is efficient, but does typically not reveal if (or how) ligands may interfere with its functional properties. For this a biochemical/functional assay is required. But for proteins whose function is dependent on a conformational change, such assays are typically complex or have low throughput. Here we have explored a high-throughput second-harmonic generation (SHG) biosensor to detect fragments that induce conformational changes upon binding to a protein in real time and identify dynamic regions. Multiwell plate format SHG assays were developed for wild-type and six engineered single-cysteine mutants of acetyl choline binding protein (AChBP), a homologue to ligand gated ion channels (LGICs). They were conjugated with second harmonic-active labels via amine or maleimide coupling. To validate the assay, it was confirmed that the conformational changes induced in AChBP by nicotinic acetyl choline receptor (nAChR) agonists and antagonists were qualitatively different. A 1056 fragment library was subsequently screened against all variants and conformational modulators of AChBP were successfully identified, with hit rates from 9–22%, depending on the AChBP variant. A subset of four hits was selected for orthogonal validation and structural analysis. A time-resolved grating-coupled interferometry-based biosensor assay confirmed the interaction to be a reversible 1-step 1 : 1 interaction, and provided estimates of affinities and interaction kinetic rate constants (KD = 0.28–63 μM, ka = 0.1–6 μM−1 s−1, kd = 1 s−1). X-ray crystallography of two of the fragments confirmed their binding at a previously described conformationally dynamic site, corresponding to the regulatory site of LGICs. These results reveal that SHG has the sensitivity to identify fragments that induce conformational changes in a protein. A selection of fragment hits with a response profile different to known LGIC regulators was characterized and confirmed to bind to dynamic regions of the protein.

    Place, publisher, year, edition, pages
    Royal Society of Chemistry, 2021
    National Category
    Biophysics
    Identifiers
    urn:nbn:se:uu:diva-429945 (URN)10.1039/D0RA09844B (DOI)000619136600042 ()
    Funder
    EU, Horizon 2020, 675899
    Note

    Title in thesis list of papers: Discovery of fragments targeting dynamic proteins using second-harmonic generation

    Available from: 2021-01-07 Created: 2021-01-07 Last updated: 2024-01-15Bibliographically approved
    6. Elucidating the regulation of ligand gated ion channels via biophysical studies of ligand-induced conformational dynamics of acetylcholine binding proteins
    Open this publication in new window or tab >>Elucidating the regulation of ligand gated ion channels via biophysical studies of ligand-induced conformational dynamics of acetylcholine binding proteins
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biophysics
    Identifiers
    urn:nbn:se:uu:diva-508763 (URN)
    Available from: 2023-08-08 Created: 2023-08-08 Last updated: 2023-08-08
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  • 41. Chapman, Henry N.
    et al.
    Caleman, Carl
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Molecular and condensed matter physics.
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Molecular and condensed matter physics. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Diffraction before destruction2014In: Philosophical Transactions of the Royal Society of London. Biological Sciences, ISSN 0962-8436, E-ISSN 1471-2970, Vol. 369, no 1647, p. 20130313-Article in journal (Refereed)
    Abstract [en]

    X-ray free-electron lasers have opened up the possibility of structure determination of protein crystals at room temperature, free of radiation damage. The femtosecond-duration pulses of these sources enable diffraction signals to be collected from samples at doses of 1000 MGy or higher. The sample is vaporized by the intense pulse, but not before the scattering that gives rise to the diffraction pattern takes place. Consequently, only a single flash diffraction pattern can be recorded from a crystal, giving rise to the method of serial crystallography where tens of thousands of patterns are collected from individual crystals that flow across the beam and the patterns are indexed and aggregated into a set of structure factors. The high-dose tolerance and the many-crystal averaging approach allow data to be collected from much smaller crystals than have been examined at synchrotron radiation facilities, even from radiation-sensitive samples. Here, we review the interaction of intense femtosecond X-ray pulses with materials and discuss the implications for structure determination. We identify various dose regimes and conclude that the strongest achievable signals for a given sample are attained at the highest possible dose rates, from highest possible pulse intensities.

  • 42.
    Cheah, Mun Hon
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Zhang, Miao
    Humboldt Univ, Inst Biol, D-10115 Berlin, Germany.
    Shevela, Dmitry
    Umea Univ, Chem Biol Ctr, Dept Chem, S-90187 Umea, Sweden.
    Mamedov, Fikret
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Zouni, Athina
    Humboldt Univ, Inst Biol, D-10115 Berlin, Germany.
    Messinger, Johannes
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Umea Univ, Chem Biol Ctr, Dept Chem, S-90187 Umea, Sweden.
    Assessment of the manganese cluster's oxidation state via photoactivation of photosystem II microcrystals2020In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 117, no 1, p. 141-145Article in journal (Refereed)
    Abstract [en]

    Knowledge of the manganese oxidation states of the oxygenevolving Mn4CaO5 cluster in photosystem II (PSII) is crucial toward understanding the mechanism of biological water oxidation. There is a 4 decade long debate on this topic that historically originates from the observation of a multiline electron paramagnetic resonance (EPR) signal with effective total spin of S = 1/2 in the singly oxidized S-2 state of this cluster. This signal implies an overall oxidation state of either Mn(III)(3)Mn(IV) or Mn(III)Mn(IV)(3) for the S-2 state. These 2 competing assignments are commonly known as "low oxidation (LO)" and "high oxidation (HO)" models of the Mn4CaO5 cluster. Recent advanced EPR and Mn K-edge X-ray spectroscopy studies converge upon the HO model. However, doubts about these assignments have been voiced, fueled especially by studies counting the number of flash-driven electron removals required for the assembly of an active Mn4CaO5 cluster starting from Mn(II) and Mnfree PSII. This process, known as photoactivation, appeared to support the LO model since the first oxygen is reported to evolve already after 7 flashes. In this study, we improved the quantum yield and sensitivity of the photoactivation experiment by employing PSII microcrystals that retained all protein subunits after complete manganese removal and by oxygen detection via a custom built thin-layer cell connected to a membrane inlet mass spectrometer. We demonstrate that 9 flashes by a nanosecond laser are required for the production of the first oxygen, which proves that the HO model provides the correct description of the Mn4CaO5 cluster's oxidation states.

  • 43. Cojoc, Gheorghe
    et al.
    Florescu, Ana-Maria
    Krull, Alexander
    Klemm, Anna H
    Pavin, Nenad
    Jülicher, Frank
    Tolić, Iva M
    Paired arrangement of kinetochores together with microtubule pivoting and dynamics drive kinetochore capture in meiosis I.2016In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 25736Article in journal (Refereed)
    Abstract [en]

    Kinetochores are protein complexes on the chromosomes, whose function as linkers between spindle microtubules and chromosomes is crucial for proper cell division. The mechanisms that facilitate kinetochore capture by microtubules are still unclear. In the present study, we combine experiments and theory to explore the mechanisms of kinetochore capture at the onset of meiosis I in fission yeast. We show that kinetochores on homologous chromosomes move together, microtubules are dynamic and pivot around the spindle pole, and the average capture time is 3-4 minutes. Our theory describes paired kinetochores on homologous chromosomes as a single object, as well as angular movement of microtubules and their dynamics. For the experimentally measured parameters, the model reproduces the measured capture kinetics and shows that the paired configuration of kinetochores accelerates capture, whereas microtubule pivoting and dynamics have a smaller contribution. Kinetochore pairing may be a general feature that increases capture efficiency in meiotic cells.

  • 44.
    Czapla-Masztafiak, Joanna
    et al.
    Paul Scherrer Inst, Villigen, Switzerland.;Polish Acad Sci, Inst Nucl Phys, Krakow, Poland..
    Szlachetko, Jakub
    Paul Scherrer Inst, Villigen, Switzerland.;Jan Kochanowski Univ Humanities & Sci, Inst Phys, Kielce, Poland..
    Milne, Christopher J.
    Paul Scherrer Inst, Villigen, Switzerland..
    Lipiec, Ewelina
    Sa, Jacinto
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry. Polish Acad Sci, Inst Phys Chem, Warsaw, Poland..
    Penfold, Thomas J.
    Newcastle Univ, Dept Chem, Bedson Bldg, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, England..
    Huthwelker, Thomas
    Paul Scherrer Inst, Villigen, Switzerland..
    Borca, Camelia
    Paul Scherrer Inst, Villigen, Switzerland..
    Abela, Rafael
    Paul Scherrer Inst, Villigen, Switzerland..
    Kwiatek, Wojciech M.
    Polish Acad Sci, Inst Nucl Phys, Krakow, Poland..
    Investigating DNA Radiation Damage Using X-Ray Absorption Spectroscopy2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 6, p. 1304-1311Article in journal (Refereed)
    Abstract [en]

    The biological influence of radiation on living matter has been studied for years; however, several questions about the detailed mechanism of radiation damage formation remain largely unanswered. Among all biomolecules exposed to radiation, DNA plays an important role because any damage to its molecular structure can affect the whole cell and may lead to chromosomal rearrangements resulting in genomic instability or cell death. To identify and characterize damage induced in the DNA sugar-phosphate backbone, in this work we performed x-ray absorption spectroscopy at the P K-edge on DNA irradiated with either UVA light or protons. By combining the experimental results with theoretical calculations, we were able to establish the types and relative ratio of lesions produced by both UVA and protons around the phosphorus atoms in DNA.

  • 45.
    Dahlberg, Tobias
    et al.
    Umeå Univ, Dept Phys, Umeå, Sweden..
    Malyshev, Dmitry
    Umeå Univ, Dept Phys, Umeå, Sweden..
    Andersson, Per Ola
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science and Engineering, Applied Material Science. Swedish Def Res Agcy FOI, Umeå, Sweden.
    Andersson, Magnus
    Umeå Univ, Dept Phys, Umeå, Sweden..
    Biophysical Fingerprinting of Single Bacterial Spores using Laser Raman Optical Tweezers2020In: Chemical, biological, radiological, nuclear, and explosives (CBRNE) sensing XXI / [ed] Guicheteau, JA Howle, CR, SPIE-Intl Soc Optical Eng , 2020, article id 114160IConference paper (Refereed)
    Abstract [en]

    Spore-forming bacteria that cause diseases pose a danger in our society. When in spore form, bacteria can survive high temperatures and resist a plethora of disinfection chemicals. Effective disinfection approaches are thus critical. Since a population of bacterial spores is heterogeneous in many aspects, single spore analyzing methods are suitable when heterogeneous information cannot be neglected. We present in this work a high-resolution Laser Raman optical tweezers that can trap single spores and characterize their Raman spectra. We first evaluate our system by measuring Raman spectra of spores, and purified DNA and DPA. Thereafter, we expose Bacillus thuringiensis spores to peracetic acid, chlorine dioxide, and sodium hypochlorite, which are common disinfection chemicals. The data reveals how these agents change the constitutes of a spore over time, thus improving on the mode of action of these disinfection chemicals.

  • 46.
    Danielsson, Emma
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Towards a better understanding of protein structures: assessing the sulfur bridge in Cystine through photofragmentation2020Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    This work aims to investigate the fragmentation of an ionized Cystine molecule, as simulated in the framework of molecular dynamics and quantum mechanics. Cystine is viewed as a model system for larger sets of peptides -- ultimately contributing to the understanding of protein photofragmentation, which is crucial for determining the structure of a protein using new methods. The analysis software was written in Python, partly in conjunction with another student. The photofragmentation of the molecule is analyzed in terms of bond integrity versus time and mass-to-charge ratios for the resulting fragments. Generally, the molecule disintegrates into more and smaller fragments the higher the degree of ionization is.

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  • 47. Danielsson, Ravi
    et al.
    Albertsson, Per-Åke
    Mamedov, Fikret
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
    Styring, Stenbjörn
    Quantification of photosystem I and II in different parts of the thylakoid membrane2004In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1608, p. 53-61Article in journal (Refereed)
  • 48. D'Arcy, Allan
    et al.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Cowan-Jacob, Sandra W.
    Marsh, May
    Microseed matrix screening for optimization in protein crystallization: what have we learned?2014In: Acta Crystallographica Section F: Structural Biology Communications, E-ISSN 2053-230X, Vol. 70, p. 1117-1126Article in journal (Refereed)
    Abstract [en]

    Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time-and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.

  • 49.
    Daurer, Benedikt J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Algorithms for Coherent Diffractive Imaging with X-ray Lasers2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Coherent diffractive imaging (CDI) has become a very popular technique over the past two decades. CDI is a "lensless" imaging method which replaces the objective lens of a conventional microscope by a computational image reconstruction procedure. Its increase in popularity came together with the development of X-ray free-electron lasers (XFELs) which produce extremely bright and coherent X-rays. By facilitating these unique properties, CDI enables structure determination of non-crystalline samples at nanometre resolution and has many applications in structural biology, material science and X-ray optics among others. This work focuses on two specific CDI techniques, flash X-ray diffractive imaging (FXI) on biological samples and X-ray ptychography.

    While the first FXI demonstrations using soft X-rays have been quite promising, they also revealed remaining technical challenges. FXI becomes even more demanding when approaching shorter wavelengths to allow subnanometre resolution imaging. We described one of the first FXI experiments using hard X-rays and characterized the most critical components of such an experiment, namely the properties of X-ray focus, sample delivery and detectors. Based on our findings, we discussed experimental and computational strategies for FXI to overcome its current difficulties and reach its full potential. We deposited the data in the Coherent X-ray Database (CXIDB) and made our data analysis code available in a public repository. We developed algorithms targeted towards the needs of FXI experiments and implemented a software package which enables the analysis of diffraction data in real time.

    X-ray ptychography has developed into a very useful tool for quantitative imaging of complex materials and has found applications in many areas. However, it involves a computational reconstruction step which can be slow. Therefore, we developed a fast GPU-based ptychographic solver and combined it with a framework for real-time data processing which already starts the ptychographic reconstruction process while data is still being collected. This provides immediate feedback to the user and allows high-throughput ptychographic imaging.

    Finally, we have used ptychographic imaging as a method to study the wavefront of a focused XFEL beam under typical FXI conditions. 

    We are convinced that this work on developing strategies and algorithms for FXI and ptychography is a valuable contribution to the development of coherent diffractive imaging. 

    List of papers
    1. Experimental strategies for imaging bioparticles with femtosecond hard X-ray pulses
    Open this publication in new window or tab >>Experimental strategies for imaging bioparticles with femtosecond hard X-ray pulses
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    2017 (English)In: IUCrJ, E-ISSN 2052-2525, Vol. 4, p. 251-262Article in journal (Refereed) Published
    National Category
    Biophysics
    Identifiers
    urn:nbn:se:uu:diva-323439 (URN)10.1107/S2052252517003591 (DOI)000400460500008 ()28512572 (PubMedID)
    Projects
    eSSENCE
    Available from: 2017-04-07 Created: 2017-11-14 Last updated: 2023-10-30Bibliographically approved
    2. Hummingbird: monitoring and analyzing flash X-ray imaging experiments in real time
    Open this publication in new window or tab >>Hummingbird: monitoring and analyzing flash X-ray imaging experiments in real time
    2016 (English)In: Journal of applied crystallography, ISSN 0021-8898, E-ISSN 1600-5767, Vol. 49, p. 1042-1047Article in journal (Refereed) Published
    National Category
    Biophysics Software Engineering
    Identifiers
    urn:nbn:se:uu:diva-287197 (URN)10.1107/S1600576716005926 (DOI)000377020600036 ()
    Projects
    eSSENCE
    Available from: 2016-04-18 Created: 2016-04-22 Last updated: 2018-01-10Bibliographically approved
    3. SHARP: a distributed GPU-based ptychographic solver
    Open this publication in new window or tab >>SHARP: a distributed GPU-based ptychographic solver
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    2016 (English)In: Journal of applied crystallography, ISSN 0021-8898, E-ISSN 1600-5767, Vol. 49, p. 1245-1252Article in journal (Refereed) Published
    Abstract [en]

    Ever brighter light sources, fast parallel detectors and advances in phase retrieval methods have made ptychography a practical and popular imaging technique. Compared to previous techniques, ptychography provides superior robustness and resolution at the expense of more advanced and time-consuming data analysis. By taking advantage of massively parallel architectures, high-throughput processing can expedite this analysis and provide microscopists with immediate feedback. These advances allow real-time imaging at wavelength-limited resolution, coupled with a large field of view. This article describes a set of algorithmic and computational methodologies used at the Advanced Light Source and US Department of Energy light sources. These are packaged as a CUDA-based software environment named SHARP (http://camera.lbl.gov/sharp), aimed at providing state-of-the-art high-throughput ptychography reconstructions for the coming era of diffraction-limited light sources.

    Keywords
    coherent X-ray diffractive imaging, ptychography, nanoscience, X-ray microscopy, phase-contrast X-ray imaging
    National Category
    Atom and Molecular Physics and Optics
    Identifiers
    urn:nbn:se:uu:diva-300178 (URN)10.1107/S1600576716008074 (DOI)000382755900015 ()
    Funder
    Swedish Research CouncilSwedish Foundation for Strategic Research
    Available from: 2016-08-04 Created: 2016-08-04 Last updated: 2017-10-24
    4. Nanosurveyor: a framework for real-time data processing
    Open this publication in new window or tab >>Nanosurveyor: a framework for real-time data processing
    Show others...
    2017 (English)In: Advanced Structural and Chemical Imaging, ISSN 2198-0926, Vol. 3, no 7Article in journal (Refereed) Published
    Abstract [en]

    Background: The ever improving brightness of accelerator based sources is enabling novel observations and discov-eries with faster frame rates, larger fields of view, higher resolution, and higher dimensionality.

    Results: Here we present an integrated software/algorithmic framework designed to capitalize on high-throughput experiments through efficient kernels, load-balanced workflows, which are scalable in design. We describe the streamlined processing pipeline of ptychography data analysis.

    Conclusions: The pipeline provides throughput, compression, and resolution as well as rapid feedback to the micro-scope operators

    Keywords
    Streaming, Ptychography
    National Category
    Computer Sciences
    Identifiers
    urn:nbn:se:uu:diva-317013 (URN)10.1186/s40679-017-0039-0 (DOI)000411110400001 ()28261545 (PubMedID)
    Funder
    Swedish Research CouncilSwedish Foundation for Strategic Research
    Available from: 2017-03-08 Created: 2017-03-08 Last updated: 2023-10-30Bibliographically approved
    5. Wavefront sensing of individual XFEL pulses using ptychography
    Open this publication in new window or tab >>Wavefront sensing of individual XFEL pulses using ptychography
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    The characterization of the wavefront dynamics is important for many X-ray free-electron laser (XFEL) experiments, in particular for coherent diffractive imaging (CDI), as the reconstructed image is always the product of the incoming wavefront with the object. An accurate understanding of the wavefront is also important for any experiment wishing to achieve peak power densities, making use of the tightest possible focal spots. With the use of ptychography we demonstrate high-resolution imaging of the Linac Coherent Light Source (LCLS) beam focused at the endstation for Atomic, Molecular and Optical (AMO) experiments, including its phase and intensity at every plane along its propagation axis, for each individual pulse. Using a mixed-state approach, we have reconstructed the most dominant beam components that constitute an ensemble of pulses, and from the reconstructed components determined their respective contribution in each of the individual pulses. This enabled us to obtain complete wavefront information about each individual pulse. We hope that our findings aid interpretation of data from past and future LCLS experiments and we propose this method to be used routinely for XFEL beam diagnostics. 

    National Category
    Atom and Molecular Physics and Optics
    Identifiers
    urn:nbn:se:uu:diva-329011 (URN)
    Available from: 2017-10-24 Created: 2017-10-24 Last updated: 2017-10-24
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  • 50.
    Daurer, Benedikt J.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hantke, Max F.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Nettelblad, Carl
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computational Science. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Maia, Filipe R. N. C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hummingbird: monitoring and analyzing flash X-ray imaging experiments in real time2016In: Journal of applied crystallography, ISSN 0021-8898, E-ISSN 1600-5767, Vol. 49, p. 1042-1047Article in journal (Refereed)
1234567 1 - 50 of 329
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