IUCrJ

Inositol monophosphatase (IMPase) is inhibited by lithium, the most efficacious treatment for bipolar disorder. Several therapies have been approved, or are going through clinical trials, aimed at the replacement of lithium in the treatment of bipolar disorder. One candidate small molecule is ebselen, a selenium-containing antioxidant, which has been demonstrated to produce lithium-like effects, both in a murine model and in clinical trials. Here we present the crystallization and first structure of human IMPase covalently complexed with ebselen, a 1.47[A] crystal structure (PDB entry 6ZK0). In the human-IMPase-complex ebselen, in a ring opened conformation, is covalently attached to Cys141, a residue located away from the active site. IMPase is a dimeric enzyme and, in the crystal structure, two adjacent dimers share four ebselen molecules, creating a tetramer with [~]222 symmetry. In the crystal structure presented in this publication, the active site in the tetramer is still accessible, suggesting that ebselen may function as an allosteric inhibitor, or may block the binding of partner proteins. SynopsisHere we present a 1.47[A] crystal structure of human inositol monophosphatase (IMPase) bound to the inhibitor ebselen (PDB entry 6ZK0). In the structure, ebselen forms a seleno-sulfide bond with cysteine 141 and ebselen-mediated contacts between two dimers give a [~]222 tetramer.

A favorable signal-to-noise ratio is essential for obtaining high-quality diffraction data in macromolecular electron crystallography. Inelastic scattering contributes significantly to the noise, reducing contrast between diffraction peaks and background, which complicates peak detection and compromises the accuracy of intensity integration. Energy filtering mitigates these challenges and enhances diffraction data quality by removing the inelastically scattered electrons, leading to reduced background noise and sharper Bragg peaks. Previously, we reported a substantial improvement in MicroED data quality and resolution with energy filtering. Here, we systematically evaluate the impact of different energy filter slit widths for optimal MicroED data collection. Data from proteinase K lamellae were collected using the 5, 10, and 20 eV energy filter slit widths. Our results show that the narrowest slit widths result in a stronger diffraction signal with lower background noise, improving the precision of the intensity measurements which resulted in better structural models. Our findings provide insights into the optimization of energy filter slit settings that, when paired with direct electron detection, enhance MicroED data collection strategies in MicroED by improving the signal-to-noise ratio, supporting higher quality data and ultimately enabling more precise structure determination.

The combination of high sensitivity and rapid readout makes it possible for electron-counting detectors to record cryogenic electron microscopy data faster and more accurately without increasing the exposure. This is especially useful for MicroED of macromolecular crystals where the strength of the diffracted signal at high resolution is comparable to the surrounding background. The ability to decrease the exposure also alleviates concerns about radiation damage which limits the information that can be recovered from a diffraction measurement. However, the dynamic range of electron-counting detectors requires careful data collection to avoid errors from coincidence loss. Nevertheless, these detectors are increasingly deployed in cryo-EM facilities, and several have been successfully used for MicroED. Provided coincidence loss can be minimized, electron-counting detectors bring high potential rewards.

X-ray crystallography is a cornerstone of biochemistry. Traditional freezing of protein crystals to cryo-temperatures mitigates X-ray damage and facilitates crystal handling but provides an incomplete window into the ensemble of conformations at the heart of protein function and energetics. Room temperature (RT) X-ray crystallography provides more extensive ensemble information, and recent developments allow conformational heterogeneity, the experimental manifestation of ensembles, to be extracted from single crystal data. However, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical question, we obtained increasingly X-ray-damaged high-resolution datasets (1.02-1.52 [A]) from single thaumatin, proteinase K, and lysozyme crystals. Heterogeneity analyses indicated a modest increase in conformational disorder with X-ray damage. Nevertheless, these effects do not alter overall conclusions and can be minimized by limiting the extent of X-ray damage or eliminated by extrapolation to obtain heterogeneity information free from X-ray damage effects. To compare these effects to damage at cryo temperature and to learn more about damage and heterogeneity in cryo-cooled crystals, we carried out an analogous analysis of increasingly damaged proteinase K cryo datasets (0.9-1.16 [A]). We found X-ray damage-associated heterogeneity changes that were not observed at RT. This observation and the scarcity of reported X-ray doses and damage extent render it difficult to distinguish real from artifactual conformations, including those occurring as a function of temperature. The ability to aquire reliable heterogeneity information from single crystals at RT provides strong motivation for further development and routine implementation of RT X-ray crystallography to obtain conformational ensemble information. SignificanceX-ray crystallography has allowed biologists to visualize the proteins that carry out complex biological processes and has provided powerful insights into how these molecules function. Our next level of understanding requires information about the ensemble of conformations that is at the heart of protein function and energetics. Prior results have shown that room temperature (RT) X-ray crystallography provides extensive ensemble information, but are subject to extenstive X-ray damage. We found that ensemble information with little or no effects from X-ray damage can be collected at RT. We also found that damage effects may be more prevalent than recognized in structures obtained under current standard cryogenic conditions. RT X-ray crystallography can be routinely implemented to obtain needed information about conformational ensembles.

Single particle Cryo-Electron microscopy (Cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution at which individual atoms, including hydrogen atoms, can be resolved. Thus Cryo-EM allows not only unprecedented detail regarding the structural architecture of complexes but also a better understanding surrounding their chemical states. In this study we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5[A] or better which so far has only been achieved for the iron carrier ferritin. Using state of the art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42[A] map of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48-hour microscope session. In addition to water molecules and ligands involved in AaLS function, we can observe positive density for [~]50% of hydrogen atoms. A small improvement in resolution was achieved by Ewald sphere correction which was expected to limit the resolution to [~]1.5[A] for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.

Single particle cryogenic electron microscopy (cryo-EM) as a structural biology methodology has become increasingly attractive and accessible to investigators in both academia and industry as this ever-advancing technology enables successful structural determination of a wide range of protein and nucleic acid targets. Although data for many high resolution cryo-EM structures are still obtained using a 300 kV cryogenic transmission electron microscope (cryo-TEM), a modern 200 kV cryo-TEM equipped with an advanced direct electron detector and energy filter is a cost-effective choice for most single particle applications, routinely achieving sub 3 angstrom ([A]) resolution. Here, we systematically evaluate performance of one such high-end configuration - a 200 kV Glacios microscope coupled with a Falcon 4 direct electron detector and Selectris energy filter (Glacios-F4-S). First, we evaluated data quality on the standard benchmarking sample, rabbit muscle aldolase, using three of the most frequently used cryo-EM data collection software: SerialEM, Leginon and EPU, and found that - despite sample heterogeneity - all final reconstructions yield same overall resolutions of 2.6 [A] and map quality when using either of the three software. Furthermore, comparison between Glacios-F4-S and a 300 kV cryo-TEM (Titan Krios with Falcon 4) revealed nominal resolution differences in overall reconstructions of a reconstituted human nucleosome core particle, achieving 2.8 and 2.5 [A], respectively. Finally, we performed comparative data analysis on the human RAD51 paralog complex, BCDX2, a four-protein complex of approximately 150 kilodaltons, and found that a small dataset ([≤]1,000 micrographs) was sufficient to generate a 3.3 [A] reconstruction, with sufficient detail to resolve co-bound ligands, AMP-PNP and Mg+2. In summary, this study provides evidence that the Glacios-F4-S operates equally well with all standard data collection software, and is sufficient to obtain high resolution structural information of novel macromolecular complexes, readily acquiring single particle data rivaling that of 300 kV cryo-TEMs.

While cryo-EM with modern direct electron detectors has proven incredibly powerful, becoming a dominant technique in structural biology, the analysis of cryo-EM images is significantly complicated by their exceptionally low signal-to-noise ratio, limiting the accuracy of the parameterisation of the physical models required for successful classification and reconstruction. Micrographs from modern direct electron detectors are typically collected as dose-fractionated multi-frame movies to allow the recording of separated individual electron impacts. These detectors improve electron detection and allow for both inter-frame motion correction, and dose-dependent image filtering, lessening the overall impact of effects deleterious to the recovery of high-resolution information. In this study we measured the information content at each spatial frequency in cryo-EM movies as it accrues during the course of an exposure. We show that, as well as correction for motion and radiation damage, the use of the information within movies allows substantially improved direct estimation of the remaining key image parameters required for accurate 3D reconstruction: the image CTF and spectral SNR. We are developing "CARYON" {insert contrived acronym here}, as a LAFTER-family filter for cryo-EM movies based upon such measurements. CARYON is intended to provide the best parameter estimation and filtration possible for a single complete, or large sub-section from a, movie micrograph without the use of a previously refined density. We demonstrate its utility in both single-particle and tomographic cryo-EM data processing.

The SARS coronavirus 2 main protease 3CLpro tailor cuts various essential virus proteins out of long poly-protein translated from the virus RNA. If the 3CLpro is inhibited, the functional virus proteins cannot form and the virus cannot replicate and assemble. Any compound that inhibits the 3CLpro is therefore a potential drug to end the pandemic. Here we show that the diffraction power of 3CLpro crystals is effectively destroyed by Ebselen. It appears that Ebselen may be a widely available, relatively cost effective way to eliminate the SARS coronavirus 2.

Cryogenic electron microscopy (cryoEM) is now routinely used for high-resolution structure determination of biological macromolecules. However, many biological specimens exhibit varying degrees of preferred orientation on cryoEM grids, resulting in uneven sampling of three-dimensional Fourier space. This orientation bias produces anisotropic reconstruction artifacts and, in severe cases, can exacerbate particle misalignment during iterative refinement, thereby limiting the success rate of near-atomic resolution cryoEM structure determination. This protocol provides a practical guide for applying spIsoNet, a self-supervised deep-learning method, to mitigate preferred-orientation issues in cryoEM reconstructions. We describe two complementary workflows: (1) map Anisotropy Correction to correct anisotropic artifacts of cryoEM maps and (2) particle Misalignment Correction, which integrates spIsoNet with RELION external reconstruction to improve particle-pose estimation. We demonstrate these workflows using two influenza hemagglutinin (HA) trimer datasets representing moderate and severe degrees of preferred-orientation bias. The protocol includes installation instructions, parameter-selection guidance, quality-control checkpoints and troubleshooting advice, and can typically be completed in ~7 hours on a workstation equipped with four NVIDIA A100 GPUs. Together, these workflows provide step-by-step guidance for using the open-source spIsoNet software to mitigate the preferred-orientation problem directly from experimental data.

Room-temperature (RT) X-ray diffraction experiments enable us to investigate protein dynamics, efficiently probe fragment binding, and perform time-resolved crystallography experiments. The Versatile Macromolecular Crystallography in-situ (VMXi) beamline at Diamond Light Source (DLS) in the United Kingdom specializes in the collection of RT X-ray diffraction data in situ directly from crystallization trays without any manipulation of protein crystals, improving crystal integrity for fragile crystals. While many X-ray sources are now equipped to grow crystals on site for in-situ experiments, to date there has been no comprehensive analysis that we are aware of on the effect of shipping crystals on plates at ambient temperature for RT data collection, while the equivalent methodology for cryo-cooled crystals is well established. Here we examine the impact of shipping on crystals grown on MiTeGen In Situ-1 plates at the University of Buffalo Hauptman Woodward Research Institute (UB-HWI) in Buffalo, NY, United States transatlantic to DLS in Didcot, United Kingdom. We utilized the Stanford Synchrotron Radiation Lightsource (SSRL) Blue Box Thermal Shipper (Blue Box), which can maintain temperature for up to 168 hours, to ship crystallization plates at room temperature from UB-HWI to DLS. We hypothesized that long-distance shipping might compromise data quality through mechanical stress or temperature fluctuations. Instead, we found that room-temperature data collected at VMXi showed no significant differences for crystals set up at UB-HWI and shipped relative to crystals set up on site in the UK. High-resolution structures were successfully determined for all proteins in the study, demonstrating that long-distance shipment of crystals at non-cryogenic temperatures is feasible without compromising diffraction quality. This study provides a proof-of-concept workflow for expanding access to room-temperature crystallography worldwide, enabling more researchers to leverage cutting-edge techniques without needing to grow crystals on site.

Atomic resolution in cryo-electron microscopy was first demonstrated six years ago. This was accomplished using 300 kV electron microscopes equipped with new hardware that provided narrower energy spread, aberration correction, and energy filtering. Here, we report the achievement of 1.24 [A] atomic resolution on an upgraded 200 kV electron microscope featuring a cold field emission gun, a high-resolution objective lens polepiece, and an energy filter. These components transform the instrument into a cost-effective single particle cryo-EM platform with performance comparable to that of significantly more expensive 300 kV systems. The microscope can also be operated at 100 kV and by using a high-speed hybrid-pixel detector we were able to reach sub-2 [A] resolution.

Accurate determination of the image pixel size is critical for quantitative cryo-electron microscopy analyses, yet existing calibration methods remain under-utilized because installation barriers and workflow complexity discourage routine adoption. To fill in this gap, a web-based application, WebCalEM, was developed to transform specialized calibration procedures into an accessible routine practice. Micrographs of any specimen with a known crystalline lattice, such as gold or graphene-oxide, are uploaded through a standard browser, processed entirely client-side, and analyzed with real-time visualization and downloadable statistical outputs. The application is delivered as a single self-contained HTML file that runs in any modern web browser without server-side computation, a configuration well suited to isolated core-facility microscope workstations. Cross-standard consistency between gold and graphene-oxide measurements across two microscopes and ten magnification settings yields a Bland-Altman bias of -0.005% of nominal with 95% limits of agreement of [-0.30%, +0.29%]. By delivering this workflow with no local installation, WebCalEM lowers the practical barrier to documented per-dataset magnification calibration in routine cryo-EM operation. SynopsisWebCalEM is a browser-based, install-free application that performs routine cryo-EM pixel-size calibration directly from gold or graphene-oxide reflections in standard sample-support grids using sub-pixel Fourier-space peak localization; it reproduces the precision of established command-line calibration tools while removing the installation barrier and supporting retrospective per-region calibration on archived datasets.

Serial femtosecond crystallography (SFX) is increasingly employed to determine protein structures and study conformational dynamics under physiological conditions, but the effects of sample preparation and delivery on crystallized macromolecules remain poorly understood. Here, we report the analysis of soybean lipoxygenase-1 (SLO) microcrystals collected by SFX at the Linac Coherent Light Source. During data analysis, we observed unexpected polymorphism in SLOs unit-cell parameters, arising from two compounding factors: indexing ambiguities caused by the pseudo-tetragonal symmetry of the SLO crystal lattice, and true non-isomorphism between individual crystals driven by differential hydration of microcrystals embedded in a hydroxyethylcellulose carrier medium. By combining unit-cell clustering with systematic reindexing, we resolved two distinct polymorphs and determined independent structures from a single experiment. Although the two structures show little difference in the average atomic coordinates (average all-atom RMSD of 0.34 [A]), an approximately 8.5% difference in crystal solvent content produces measurable differences in crystal contacts and conformational flexibility. The more hydrated (large-cell) polymorph exhibits greater inter-domain flexibility and harmonic disorder in hydrophobic core residues belonging to the catalytic network of the enzyme. In the dehydrated (small-cell) polymorph, these same residues adopt discrete alternative conformations resolvable in the electron density. Our results demonstrate that sample delivery conditions in serial crystallography can significantly modulate the apparent conformational landscape of crystallized proteins, with direct implications for the interpretation of protein dynamics from SFX data. SynopsisUsing soybean lipoxygenase-1 (SLO) as a model system, we show that the carrier media used for sample delivery in SFX experiments can alter the solvent content of protein microcrystals, producing distinct crystal polymorphs within a single experimental sample. Although the average atomic coordinates are minimally perturbed, a detailed analysis of crystallographic displacement parameters reveals that conformational flexibility is sensitive to sample delivery conditions, a consideration of broad relevance for time-resolved SFX experiments that aim to capture functionally important protein motions.

The advent of serial crystallography has rejuvenated and popularised room temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g., large complexes, membrane proteins, and viruses) forming fragile crystals, are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallisation. Many medically relevant samples are expressed in mammalian cell-lines, producing a meagre quantity of protein that is incompatible for batch crystallisation. This can limit the scope of serial crystallography approaches. Direct in-situ data collection from a 96-well crystallisation plate enables not only the identification of the best diffracting crystallisation condition, but also the possibility for structure determination at ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallisation plates, mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility (ESRF). We applied our iSX approach on a challenging project, Autotaxin, a therapeutic target expressed in a stable human cell-line, to determine a structure in the lowest symmetry P1 space group at 3.0 [A] resolution. Our in situ data collection strategy provided a complete dataset for structure determination, while screening various crystallisation conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallisation plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons. SynopsisThe determination of a challenging structure in the P1 space group, the lowest symmetry possible, shows how our in-situ serial crystallography approach expands the application of crystallisation plates as a robust sample delivery method.

Structure elucidation of biological macromolecules by single particle cryogenic electron microscopy (SPA cryo-EM) or cryogenic electron tomography (cryo-ET) relies on low-dose imaging on cryogenic transmission electron microscopes (cryo-TEMs). Routine microscope setup remains technically demanding and can be time-consuming, particularly for inexperienced or infrequent users. We present LowDoseWizard, a guided workflow implemented in SerialEM that enables rapid and standardised setup of cryo-TEM imaging conditions. From minimal user input, the workflow configures microscope optics, camera parameters and image shift settings for all low-dose imaging states, and guides the user through key daily alignment procedures including beam shift offset calibration, objective lens astigmatism correction and coma-free alignment. The workflow is organised into modular routines that can be executed sequentially or independently, while microscope-specific acquisition parameters are defined in editable configuration files, allowing flexible adaptation to different instruments without modification of the core scripts. Across user sessions on three microscopes at EMBL Heidelberg, the complete setup required on average less than 15 minutes. To assess whether predefined imaging conditions generated by the workflow are compatible with high-resolution data collection, we acquired apoferritin data on a 200 kV Glacios and a 300 kV Titan Krios. These datasets yielded reconstructions at 1.62 [A] and 1.09 [A] resolution, respectively, demonstrating that rapid, guided setup can support near-atomic and atomic-resolution single particle cryo-EM. LowDoseWizard lowers the barrier to robust cryo-TEM setup, reduces the time spent on routine parameter selection and alignment, and helps users focus on sample-specific aspects of data acquisition such as target selection. The workflow should be particularly valuable in shared instrumentation environments, where accessibility, reproducibility and efficient microscope use are critical.

OaPAC is a recently discovered blue-light using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report the crystal structures of the enzyme in the absence of its natural substrate determined from room temperature serial crystallography data collected at both an X-ray free electron laser and a synchrotron and we compare them with the cryo macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6 mutation in the blue-light using flavin adenosine dinucleotide domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side-chain of the critical Q48 residue. These studies pave the way for ps - ms time-resolved serial crystallography experiments at X-ray free electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well-studied ps - ms spectroscopic intermediates. SynopsisStructures of the dark-adapted state of a photoactivated adenylate cyclase are determined from serial crystallography (SX) data collected at room temperature at an X-ray free electron laser (XFEL) and a synchrotron and are compared with cryo macromolecular crystallography (MX) synchrotron structures obtained by us and others. These structures of the wild-type enzyme in combination with the cryo MX synchrotron structure of a light-sensor domain mutant provide insight into the hydrogen bond network rearrangement upon blue-light illumination and pave the way for the determination of structural intermediates of the enzyme by time-resolved SX.

Capturing meta-stable conformations of enzymes and ligand complexes demands structural snapshots beyond static crystal structures. While time-resolved serial crystallography at room temperature, offers a time-resolution down to the femto-second domain it requires large amounts of micro crystals, specialized beamlines and considerable experience. Moreover, as the majority of enzymes displays turnover-times in the millisecond domain or slower, simpler methods can provide meaningful structural insight into enzyme catalysis. Vitrification of protein crystals can trap reaction intermediates by rapid cooling to {inverted exclamation} 100 K, and has traditionally been used to gain insight into long lived reaction intermediates such as product complexes. However, manual vitrification procedures are limited to long delay times of at least several seconds and heavily suffer from operator variability. A solution to this problem is provided by automatic crystal plunging devices, such as the Spitrobot, that plunge loop-mounted protein crystals into liquid nitrogen within millisecond time-scales. Versatile means of reaction initiation can be achieved either by micro dispensing a ligand droplet, or via optical excitation of light-sensitive proteins, or via the photoactivation of caged compounds. In addition to the conceptual simplicity, another benefit of cryo-trapping is that data can be collected at conventional synchrotron beamlines, exploiting their robust high-throughput capabilities. Thus, compared to room-temperature time-resolved crystallography, users not only benefit from uncoupling sample-preparation and data-collection, but also from a reduction in the required technical expertise and ready access to radiation sources. However, as cryo-trapping crystallography explores dynamic structural changes that become only visible by the comparison of several samples, experiments have to be carefully planned to carry out the necessary controls and to avoid mis- or over-interpretation of the results. Here we describe a detailed protocol for cryo-trapping time-resolved crystallography using automated crystal-plungers that enables researchers to map enzymatic reaction coordinate pathways within the millisecond domain.

Three-dimensional electron diffraction (3D ED), also known as microcrystal electron diffraction (MicroED), is an emerging method for determining structures of submicron-sized crystals. With the development of rapid and convenient data collection protocols, acquiring dozens of datasets in a single MicroED session has become routine. A fast and automated workflow for processing, scaling and merging a large number of MicroED datasets can significantly accelerate the structure determination process. Herein, we present an XDS-based graphical user interface for automated real-time and offline batch 3D ED/MicroED data processing (AutoLEI). We illustrate the functionality of the GUI through four examples, demonstrating both offline and real-time data processing capabilities. These examples include small organic molecules, metal-organic frameworks (MOFs), and proteins, showcasing the versatility and efficiency of the GUI in various applications. SynopsisA graphical user interface for real-time and offline 3D ED/MicroED data processing by XDS was developed. The GUI aims to improve efficiency, minimize redundant data processing work, and provide users with real-time feedback during data collection.

Super resolution detector acquisition for cryo-EM has been used to improve the clarity of cryo-EM reconstructions. Recent reports have demonstrated achieving resolutions beyond the physical Nyquist limit using super resolution acquisition. Here, we demonstrate exceeding the physical Nyquist limitation by pre-processing the raw micrograph movies from "counting mode" data which has already reached physical Nyquist reconstruction resolution. To demonstrate functionality, micrograph movies of five datasets were pre-processed and demonstrate that it is possible to exceed the physical Nyquist limit via pixel doubling before motion correction. We call this "post-acquisition super resolution", or PASR. While this was originally developed for processing of giant virus datasets, where acquiring at high magnification is not always possible or desirable, it is also shown to work for smaller objects such as adeno-associated virus (AAV) and apoferritin, both of which are still high symmetry, and jack bean urease, with lower symmetry. PASR could reduce the magnification required to achieve desired resolutions, which may increase collection efficiency. PASR can also be of use for in vivo tomography and facilities with high storage demands. However, this method should only be used for data which is able to achieve the Nyquist limit without PASR pre-processing. It will not improve attained resolutions of data which does not already reach the Nyquist limit.

IntroductionProteins that adopt multiple conformations pose significant challenges in structural biology research and pharmaceutical development, as structure determination via single particle cryo-electron microscopy (cryo-EM) is often impeded by data heterogeneity. In this context, the enhanced signal-to-noise ratio of single molecule cryo-electron diffraction (simED) offers a promising alternative. However, a significant challenge in diffraction methods is the loss of phase information, which is crucial for accurate structure determination. MethodsHere, we present DiffGAN, a conditional generative adversarial network (cGAN) that estimates the missing phases at high resolution from a combination of high-resolution single particle diffraction data and low-resolution image data. ResultsFor simulated datasets, DiffGAN allows effectively determine protein structures at atomic resolution from diffraction patterns and noisy low-resolution images. DiscussionOur findings suggest that combining single particle cryo-electron diffraction with advanced generative modeling, as in DiffGAN, could revolutionize the way protein structures are determined, offering a more accurate and efficient alternative to existing methods.