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Time-resolved cryogenic electron microscopy (cryo-EM) is a powerful technique for capturingtransient molecular and viral structures during conformational changes. This method providesunique and potentially critical insights into the transient states of biomolecules, which can beinvaluable for drug development. Additionally, it offers glimpses into the pathology of viruses asthey interact with their immediate environment. In this project, a plunge freezer initially designedfor a spray-and-plunge approach in time-resolved cryo-EM [24] was modified to implement aflash-and-freeze system [3]. This modified system was tested on two different viruses: influenzaH3N2 type A and Chaetoceros tenuissimus DNA virus type II. The primary objective was tovisualize an intermediate state during the membrane fusion process between influenza A andliposomes with endosomal characteristics. Although no intermediate state was captured forinfluenza, the activation and pH reduction were successfully achieved. The study on Chaetocerostenuissimus DNA virus type II focused on potential conformational changes due to a drop in pH,rather than capturing intermediate states in a time-resolved manner. Future experiments withmore precise control over blotting, delivered intensity, and the concentration of the cagedcompound are expected to facilitate the capture and analysis of intermediate states.

The relevance of gas-phase protein structure to its solution structure is of the utmost importance in studying biomolecules by mass spectrometry. D-Amino acid substitutions within a minimal protein. Trp-cage. were used to correlate solution-phase properties as measured by circular dichroism with solution/gas-phase conformational features of protein cations probed via charge state distribution (CSD) in electrospray ionization. and gas-phase features revealed by tandem mass spectrometry (MS/MS). The gas-phase features were additionally supported by force-field molecular dynamics simulations. CD data showed that almost any single-residue D-substitution destroys the most prominent CD feature of the "native" all-L isomer, alpha-helicity. CSD was able to qualitatively assess the degree of compactness of solution-phase molecular structures. CSD results were consistent with the all-L form being the most compact in solution among all studied stereoisomers except for the D-Asn(1) isomer. D-substitutions of the aromatic Y(3), W(6) and Q(5) residues generated the largest deviations in CSD data among single amino acid substitutions. consistent with the critical role of these residues in Trp-cage stability. Electron capture dissociation of the stereoisomer dications gave an indication that some gas-phase structural features of Trp-cage are similar to those in solution. This result is supported by MDS data oil five of the studied stereoisomer dications in the gas-phase. The MDS-derived minimum-energy structures possessed more extensive hydrogen bonding than the solution-phase structure of the native form, deviating from the latter by 3-4 angstrom and were not 'inside-out' compared to native structures. MDS data could be correlated with CD data and even with ECD results. which aided in providing a long-range structural constraint for MDS. The overall conclusion is the general resemblance, despite the difference on the detailed level, of the preferred structures in both phases for the mini protein Trp-cage.

Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profiled the sequence dependence of DNA hairpin properties and Cas9-induced target DNA unwinding-rewinding dynamics. The ability to explore a large sequence space for Cas9 allowed us to identify a number of target sequences with unexpected behaviors. We envision that MUSCLE will enable the mechanistic exploration of many fundamental biological processes.

The Editors of Journal of Applied Crystallography mark the journal's 50th anniversary.

Viruses pose significant threats to human health, making it crucial to understand their structure and behavior to develop effective treatments. This study focuses on two strains of Giardia Lamblia virus (GLV) (a cat strain and human strain) a virus that infects the parasite Giardia Lamblia which in turn can infect humans or animals. Understanding viruses helps immensely with treating people who are infected as well as our ability to utilize the virus for good. The Omono River virus (OmRV) and saccharomyces cerevisae virus (ScV-L-A) is used for comparison. OmRV belongs to a group called totivirus-like viruses and ScV-L-A and GLV belong to the Totiviridae group. They have some common characteristics such as isometric virions, double-stranded RNA genomes, and 120 chemically identical subunits. What sets them apart is their hosts, viruses in the Totiviridae group only infect protozoan hosts intracellularly while OmRV only infects metazoan hosts extracellularly. GLV is interesting because it has displayed both an intra- and extracellular mode of infection. Finding out more about GLV could give us insight into viral evolution. Cryo-electron microscopy (cryoEM) is used to investigate the structure of GLV. From the cryoEM data models were created using Coot and Chimera with resolutions of 4.0 Å for the human strain and 4.2 Å for the cat strain. Found in these models were an open pore compared to the closed one of OmRV as well as lack of a C-terminal, which is used to increase stability. Signs of conserved 𝛼-helices are also found indicating their evolutionary relationship. 

Studies of materials under extreme conditions have relevance to a broad area of research, including planetary physics, fusion research, materials science, and structural biology with x-ray lasers. We study such extreme conditions and experimentally probe the interaction between ultrashort soft x-ray pulses and solid targets (metals and their deuterides) at the FLASH free-electron laser where power densities exceeding 10¹⁷ W/cm² were reached. Time-of-flight ion spectrometry and crater analysis were used to characterize the interaction. The results show the onset of saturation in the ablation process at power densities above 10¹⁶ W/cm². This effect can be linked to a transiently induced x-ray transparency in the solid by the femtosecond x-ray pulse at high power densities. The measured kinetic energies of protons and deuterons ejected from the surface reach several keV and concur with predictions from plasma-expansion models. Simulations of the interactions were performed with a nonlocal thermodynamic equilibrium code with radiation transfer. These calculations return critical depths similar to the observed crater depths and capture the transient surface transparency at higher power densities.

The first hard X-ray laser, the Linac Coherent Light Source (LCLS), produces 120 shots per second. Particles injected into the X-ray beam are hit randomly and in unknown orientations by the extremely intense X-ray pulses, where the femtosecond-duration X-ray pulses diffract from the sample before the particle structure is significantly changed even though the sample is ultimately destroyed by the deposited X-ray energy. Single particle X-ray diffraction experiments generate data at the FEL repetition rate, resulting in more than 400,000 detector readouts in an hour, the data stream during an experiment contains blank frames mixed with hits on single particles, clusters and contaminants. The diffraction signal is generally weak and it is superimposed on a low but continually fluctuating background signal, originating from photon noise in the beam line and electronic noise from the detector. Meanwhile, explosion of the sample creates fragments with a characteristic signature. Here, we describe methods based on rapid image analysis combined with ion Time-of-Flight (ToF) spectroscopy of the fragments to achieve an efficient, automated and unsupervised sorting of diffraction data. The studies described here form a basis for the development of real-time frame rejection methods, e. g. for the European XFEL, which is expected to produce 100 million pulses per hour. (C)2014 Optical Society of America

Flavin mononucleotide (FMN) belongs to the large family of flavins, ubiquitous yellow-coloured biological chromophores that contain an isoalloxazine ring system. As a cofactor in flavoproteins, it is found in various enzymes and photosensory receptors, like those featuring the light-oxygen-voltage (LOV) domain. The photocycle of FMN is triggered by blue light and proceeds via a cascade of intermediate states. In this work, we have studied isolated FMN in an aqueous solution in order to elucidate the intrinsic electronic and vibrational changes of the chromophore upon excitation. The ultrafast transitions of excited FMN were monitored through the joint use of femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption spectroscopy encompassing a time window between 0 ps and 6 ns with 50 fs time resolution. Global analysis of the obtained transient visible absorption and transient Raman spectra in combination with extensive quantum chemistry calculations identified unambiguously the singlet and triplet FMN populations and addressed solvent dynamics effects. The good agreement between the experimental and theoretical spectra facilitated the assignment of electronic transitions and vibrations. Our results represent the first steps towards more complex experiments aimed at tracking structural changes of FMN embedded in light-inducible proteins upon photoexcitation.

Intense femtosecond x-ray pulses from free-electron laser sources allow the imag-ing of individual particles in a single shot. Early experiments at the Linac CoherentLight Source (LCLS) have led to rapid progress in the field and, so far, coherentdiffractive images have been recorded from biological specimens, aerosols, andquantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLSheld a workshop to discuss the scientific and technical challenges for reaching theultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap towardreaching atomic resolution, 3D imaging at free-electron laser sources.