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We simulated Coulomb explosion dynamics due to fast ionization induced by high-intensity x-rays in six proteins that share similar atomic content and shape. We followed and projected the trajectory of the fragments onto a virtual detector, providing a unique explosion footprint. After collecting 500 explosion footprints for each protein, we utilized principal component analysis and 𝑡-distributed stochastic neighbor embedding to classify these. Results show that the classification algorithms were able to separate proteins on the basis of explosion footprints from structurally similar proteins into distinct groups. The explosion footprints, therefore, provide a unique identifier for each protein. We envision that method could be used concurrently with single-particle coherent imaging experiments to provide additional information on shape, mass, or conformation.

Collision induced unfolding is method used with ion mobility mass spectrometry to examine protein structures and their stability. Such experiments yield information about higher order protein structures, yet are unable to provide details about the underlying processes. That information can however be provided using molecular dynamics simulations. Here, we investigate the collision induced unfolding of norovirus capsid dimers from the Norwalk and Kawasaki strains by employing molecular dynamics simulations over a range of temperatures, representing different levels of activation. The dimers have highly similar structures, but the activation reveals differences in the dynamics that arises in response to the activation.

Single particle imaging of proteins in the gas phase with x-ray free-electron lasers holds great potential to study fast protein dynamics, but is currently limited by weak and noisy data. A further challenge is to discover the proteins' orientation as each protein is randomly oriented when exposed to x-rays. Algorithms such as the expand, maximize, and compress (EMC) exist that can solve the orientation problem and reconstruct the three-dimensional diffraction intensity space, given sufficient measurements. If information about orientation were known, for example, by using an electric field to orient the particles, the reconstruction would benefit and potentially reach better results. We used simulated diffraction experiments to test how the reconstructions from EMC improve with particles' orientation to a preferred axis. Our reconstructions converged to correct maps of the three-dimensional diffraction space with fewer measurements if biased orientation information was considered. Even for a moderate bias, there was still significant improvement. Biased orientations also substantially improved the results in the case of missing central information, in particular in the case of small datasets. The effects were even more significant when adding a background with 50% the strength of the averaged diffraction signal photons to the diffraction patterns, sometimes reducing the data requirement for convergence by a factor of 10. This demonstrates the usefulness of having biased orientation information in single particle imaging experiments, even for a weaker bias than what was previously known. This could be a key component in overcoming the problems with background noise that currently plague these experiments.

We describe a method to compute photon–matter interaction and atomic dynamics with x-ray lasers using a hybrid code based on classical molecular dynamics and collisional-radiative calculations. The forces between the atoms are dynamically determined based on changes to their electronic occupations and the formation of a free electron cloud created from the irradiation of photons in the x-ray spectrum. The rapid transition from neutral solid matter to dense plasma phase allows the use of screened potentials, reducing the number of non-bonded interactions. In combination with parallelization through domain decomposition, the hybrid code handles large-scale molecular dynamics and ionization. This method is applicable for large enough samples (solids, liquids, proteins, viruses, atomic clusters, and crystals) that, when exposed to an x-ray laser pulse, turn into a plasma in the first few femtoseconds of the interaction. We present four examples demonstrating the applicability of the method. We investigate the non-thermal heating and scattering of bulk water and damage-induced dynamics of a protein crystal using an x-ray pump–probe scheme. In both cases, we compare to the experimental data. For single particle imaging, we simulate the ultrafast dynamics of a methane cluster exposed to a femtosecond x-ray laser. In the context of coherent diffractive imaging, we study the fragmentation as given by an x-ray pump–probe setup to understand the evolution of radiation damage in the time range of hundreds of femtoseconds.

Copper (Cu), with its ability to exist in various oxidation states, notably Cu(I) and Cu(II), plays a crucial role in diverse biological redox reactions. This includes its involvement in pathways associated with oxidative stress in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Transmissible Spongiform Encephalopathies. This paper offers an overview of X-ray Absorption Spectroscopy (XAS) studies designed to elucidate the interactions between Cu ions and proteins or peptides associated with these neurodegenerative diseases. The emphasis lies on XAS specificity, revealing the local coordination environment, and on its sensitivity to Cu oxidation states. Furthermore, the paper focuses on XAS applications targeting the characterization of intermediate reaction states and explores the opportunities arising from recent advancements in time-resolved XAS at ultrabright synchrotron and Free Electron Laser radiation sources. Time-resolved X-ray Absorption Spectroscopy will be the camcorder for capturing atomic resolution molecular movies. The key players in these movies are Cu(I) and Cu(II) ions, which, when complexed with proteins and peptides associated with neurodegenerative diseases, can act as redox agents with potential pathogenic implications. image

Radiation therapy uses ionizing radiation to break chemical bonds in cancer cells, thereby causing DNA damage and leading to cell death. The therapeutic effectiveness can be further increased by making the tumor cells more sensitive to radiation. Here, we investigate the role of the initial halogen atom core hole on the photofragmentation dynamics of 2-bromo-5-iodo-4-nitroimidazole, a potential bifunctional radiosensitizer. Bromine and iodine atoms were included in the molecule to increase the photoionization cross-section of the radiosensitizer at higher photon energies. The fragmentation dynamics of the molecule was studied experimentally in the gas phase using photoelectron-photoion-photoion coincidence spectroscopy and computationally using Born-Oppenheimer molecular dynamics. We observed significant changes between shallow core (I 4d, Br 3d) and deep core (I 3d) ionization in fragment formation and their kinetic energies. Despite the fact, that the ions ejected after deep core ionization have higher kinetic energies, we show that in a cellular environment, the ion spread is not much larger, keeping the damage well-localized. A study on photodissociation dynamics of 2-bromo-5-iodo-nitroimidazole - a model radiosensitizer - using coincidence spectroscopy and computational methods.

- Single particle imaging using X-ray lasers is a technique aiming to capture atomic resolution structures of biomolecules in their native state. Knowing the particle's orientation during exposure is crucial for method enhancement. It has been shown that the trajectories of sulfur atoms in a Coulomb exploding lysozyme are reproducible, providing orientation information. This study explores if sulfur atom depth influences explosion trajectory. Employing a hybrid collisional-radiative/molecular dynamics model, we analyze the X-ray laser-induced dynamics of a single sulfur ion at varying depths in water. Our findings indicate that the ion spread-depth relationship depends on pulse parameters. At a photon energy of 2 keV, high-charge states are obtained, resulting in an increase of the spread with depth. However, at 8 keV photon energy, where lower charge states are obtained, the spread is essentially independent with depth. Finally, lower ion mass results in less reproducible trajectories, opening a promising route for determining protein orientation through the introduction of heavy atoms.

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm⁻¹) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

Proteins can be oriented in the gas phase using strong electric fields, which brings advantages for structure determination using X-ray free electron lasers. Both the vacuum conditions and the electric-field exposure risk damaging the protein structures. Here, we employ molecular dynamics simulations to rehydrate and relax vacuum and electric-field exposed proteins in aqueous solution, which simulates a refinement of structure models derived from oriented gas-phase proteins. We find that the impact of the strong electric fields on the protein structures is of minor importance after rehydration, compared to that of vacuum exposure and ionization in electrospraying. The structures did not fully relax back to their native structure in solution on the simulated timescales of 200 ns, but they recover several features, including native-like intra-protein contacts, which suggests that the structures remain in a state from which the fully native structure is accessible. Our fndings imply that the electric fields used in native mass spectrometry are well below a destructive level, and suggest that structures inferred from X-ray difraction from gas-phase proteins are relevant for solution and in vivo conditions, at least after in silico rehydration.

Experimental near-edge x-ray-absorption fine-structure (NEXAFS) spectra of the nitrosonium NO+ ion are presented and theoretically analyzed. While neutral NO has an open shell, the cation is a closed-shell species, which for NEXAFS leads to the simplicity of a closed-shell spectrum. Compared to neutral NO, the electrons in the cation experience a stronger Coulomb potential, which introduces a shift of the ionization potential towards higher energies, a depletion of intensity in a large interval above the pi* resonance, and a shift of the sigma* resonance from the continuum to below the ionization threshold. NEXAFS features at the nitrogen and oxygen K edges of NO+ are compared, as well as NEXAFS features at the nitrogen edges of the isoelectronic closed-shell species NO+, N2, and N2H+.