Journal of Applied Crystallography

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.

Accurate determination of state occupancies is essential for interpreting the structural heterogeneity inherent in time-resolved crystallography. However, in cases of high spatial overlap between states, as commonly observed in time-resolved crystallography data, the strong correlation between occupancy and atomic displacement parameters (ADPs) can render single point estimates from standard refinement protocols unreliable. We introduce MEROS (Multi-state Ensemble Refinement for Occupancy Statistics), a pipeline that implements an ensemble refinement approach to assess the post-refinement occupancy-ADP statistics of multiple overlapping states. MEROS utilizes a Monte Carlo sampling of the parameter space, performing independent refinements from randomized starting occupancies and ADP values to empirically characterize the convergence and uncertainty of the solution. The method is implemented as a modular Python pipeline that wraps established refinement programs, ensuring compatibility with existing workflows. We demonstrate its applicability in two case studies: a two-state ligand binding model in T4 lysozyme L99A and a four-state covalent catalysis mechanism in {beta}-lactamase CTX-M-14. MEROS provides occupancy and ADP mean values with standard deviations that directly quantify the informational content of the experimental diffraction data.

Small-angle X-ray or neutron scattering (SAXS/SANS) analysis of large unilamellar vesicles (LUVs) is often limited by high-dimensional bilayer models and the lack of dedicated, statistically rigorous workflows. Here, we introduce SAS_MoCa, an open-source Python package that integrates a compositional scattering density profile (SDP) description of lipid bilayers with a separated form factor (SFF) treatment of vesicle size and polydispersity, and couples these highly parameterized models to an adaptive thermodynamic simulated annealing algorithm formulated within a constrained Bayesian framework. SAS_MoCa enables users to incorporate quantitative prior information from, e.g., previous SAXS/SANS studies, dynamic light scattering, NMR, or molecular simulations, and returns full posterior parameter distributions, uncertainties (reported as medians and median absolute deviations) and correlations even from single SAXS curves. Validation on POPC, POPE and DMPC SAXS-only data demonstrates that the method yields reproducible structural parameters with uncertainties comparable to joint SAXS/contrast-variation SANS analyses. The modular architecture of SAS_MoCa facilitates extension to additional lipid systems and future joint SAXS/SANS or SANS-only applications.

Four-dimensional scanning transmission electron microscopy (4D-STEM) enables the acquisition of diffraction patterns at every probe position in a dense array. For imaging applications this approach offers significant benefits in terms of spatial resolution and contrast enhancement. In this work, we present the development of a synchronous scan generator integrated with SerialEM software to enable automation of complex experimental protocols such as tomography. The proposed hardware functions as an interface between SerialEM, the scan controls of the microscope, a fast annular dark-field detector, and a synchronized trigger for a pixelated detector. Our previous implementation, named SavvyScan, relied on a dedicated computer equipped with a multichannel acquisition and signal-generation cards, as well as a separate microcontroller for synchronization. Here, we report a low-cost implementation based on a Red Pitaya board, utilizing direct programming of its embedded FPGA and Linux server components. We provide detailed instructions for system installation and operation, along with practical guidance for modifying the source code. System performance is validated through oscilloscope measurements and imaging of a replica grating sample. The utility of the approach is further demonstrated by generating a 3D electron tomogram of a cryogenic sample of mitochondria from a tilt series of shadow montage projections.

Chemical imaging is a powerful branch of modern microscopy encumbered by a lack of flexible, high-throughput analysis tools. Bespoke analytical pipelines typically perform ratiometric analysis on two layers in a multispectral image to describe the relative composition of molecules in a sample. This strategy has been implemented across fields, spanning histopathology, cell biology, environmental science, and materials science. The commercialization of chemical imaging microscopes has facilitated the collection of large multispectral datasets, necessitating accessible ways to process them. This paper describes Multispectral Analysis Graphical User Interface (msaGUI), a desktop graphical user interface to analyze individual and batch datasets of multispectral images. Data is loaded as CSV, TSV, or TIFFs and processed through a user-defined sequence of modular image operations that can be flexibly combined, e.g. to reduce spectral crosstalk or background noise. After analysis, data is visualized as exportable images, histograms, and statistics. To yield publication-quality figures, outputted images are fully customizable. Written in Python with open-source libraries, the msaGUI program is packaged into an executable for Windows and Mac for a fully no-code application. Other operating systems are supported via the Python source code. In summary, msaGUI provides a rapid and user-friendly solution for analyzing and visualizing multispectral data.

Due to recent technological advances, in situ structural cell biology is becoming a high throughput microscopy technique as all the steps of the workflow, from sample preparation to data analysis, are executed faster, more reliable and more reproducible. Sample thinning by cryoFIB-SEM is an essential tool in preparing electron transparent lamellae of biological specimens suitable for further characterization by cryoET. Modern cryoFIB-SEM instruments can be operated remotely and are capable of automated and unsupervised lamellae preparation. To take full advantage of these developments they need a constant supply of LN2 to maintain cryogenic conditions inside the microscope chamber. Here, we introduce a custom automated LN2 refill system that is compatible with gas cooled cryostages, supports long-term cryoFIB-SEM operations and liberates the user from highly repetitive and manual work. We believe this solution can be utilized with other cryoSEM or cryoFIB-SEM devices requiring N2 gas-flow cooling.

Room temperature serial crystallography offers advantages over conventional cryo-crystallography, such as simplified crystal handling and the possibility to avoid potential artefacts associated with cryo-trapping. However, to be considered as an alternative for drug discovery, where compound availability may be limited and speed of structure delivery is a key factor, it suffers from several limitations. To address these challenges, we have optimized a serial crystallography workflow for ligand soaking, data collection and data processing, significantly reducing time and reagent consumption to make it a viable option for drug discovery applications, herein exemplified by crystallographic fragment screening. Our approach incorporates the use of dried-in fragment cocktails on fixed target supports, compatible with 96-well plates for crystal soaking, and an efficient data processing pipeline tailored for serial crystallography. To validate our workflow, we conducted an in-crystal fragment screen at room temperature on the protein soluble epoxide hydrolase. The screen comprised 384 compounds and resulted in identification of 40 fragment binders corresponding to a hit rate of 10.4 %. The resulting room-temperature structures are of high quality and reveal opportunities for specific interaction within the highly hydrophobic active site of soluble epoxide hydrolase. Finally, we discuss potential avenues for further workflow optimization, highlighting the future potential of this approach for drug discovery. SynopsisWe have developed a workflow that allowed us to efficiently conduct a fragment screen at room temperature using serial crystallography, of interest for future drug discovery campaigns.

Optical microscopy is fundamental to modern life-science research, yet interpreting its results requires precise modelling of point spread functions (PSFs) within complex environments. This manuscript introduces a versatile and efficient approach to wave-optical PSF calculations that extends existing frameworks by incorporating detection PSF modelling through the principle of reciprocity. Accompanying this work is a free MATLAB software package centred on a single, minimalistic core function, PlaneWaveExc.m, which utilizes a plane-wave superposition based on the Richards-Wolf model. Despite its simplicity, the framework accounts for "real-life" complexities such as systemic aberrations, arbitrary amplitude and phase modulations, and stratified media with complex-valued refractive indices. We demonstrate the softwares broad applicability through diverse case studies, including single-molecule imaging, STED microscopy, the segmented aperture of the James Webb Space Telescope, and coherent wide-field iSCAT microscopy. Each example is supported by dedicated scripts to facilitate adaptation for specific research needs.

Microscopes are essential tools for discoveries on a scale invisible to the unaided human eye. The development of immuno-fluorescence followed by molecular biology techniques and fluorescent fusion proteins have revolutionised the use of optical microscopy in bioscience. The quality of the data produced is dependent upon the sample, its preparation and the instrument used. However, instruments can degrade over time without easily visible changes to the produced images and, in turn, negatively impacts results. By testing instruments and doing comparisons between results over time and between different instruments, problems can be highlighted and corrective action can be taken. Using small fluorescent beads the point spread function (PSF) of the microscope can be recorded and the image resolution measured. Beads were prepared in a concentration matched to the field of view size and dried onto coverslips and mounted on slides. The beads were then imaged as 3D Z-stacks of sufficient size to fully enclose the PSF of the system. This data was uploaded to OMERO and processed using OMERO-metrics, an OMERO plugin developed for this purpose. This paper summarizes the development of workflows and protocols to enable this process, presents the results obtained and demonstrates the detection of significant instrument issues.

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.

Imaging the structural organization inside cells in their native state is essential for understanding how the arrangement and interactions among molecular components give rise to biological function. Fluorescence microscopy is one of the pivotal techniques that provides molecular specificity for imaging in real space, however, the technique is limited to labeled components. X-rays, on the contrary, are sensitive to electron density contrast and therefore to label-free samples, and probe structure in reciprocal space. In particular, scanning small-angle X-ray scattering (SAXS) combines information from real and reciprocal space and enables access to intact cells, owing to the high penetration power of the X-rays. Combining both imaging modalities in a synergistic manner promises powerful tools for cellular imaging, but remains challenging, because of the differing requirements the complementary methods introduce. Here we present a correlative imaging platform that integrates a modular, compact and beamline-compatible fluorescence microscope with scanning SAXS, to enable fast sequential imaging of the identical cellular regions. We developed a dedicated microfluidics flow chamber enabling measurements under hydrated, near-native conditions. We demonstrate the utility of our methodology by investigating two different relevant cellular components, i.e., thick keratin bundles in epithelial cells that contribute to cell mechanics, and force-generating actomyosin in cardiomyocytes. Employing adapted data analysis methods, we find a good agreement between the fluorescence-derived and the SAXS-derived orientation maps. This result demonstrates that the label-free approach with SAXS captures cytoskeletal organization through-out the cell, and can be directly linked to specific molecular information provided by the complementary fluorescence imaging, in a physiologically relevant cellular environment. Our work establishes a general strategy for multimodal imaging of cellular architecture and opens ways to investigate living cells under the influence of drugs and chemical manipulation experiments.

Serial femtosecond crystallography (SFX) has revolutionised structural biology by enabling direct visualization of conformational dynamics of biomacromolecules at near-physiological temperatures. However, conventional SFX sample delivery at X-ray free-electron laser (XFEL) facilities introduces a significant X-ray scattering background, which may limit the data quality and resolution. Here, we introduce an aerosol delivery-based method that drastically reduces the back-ground scattering by minimising the liquid environment surrounding the nanocrystals. We validate our method by solving the structure of Cydia pomonella granulovirus nanocrystals at 1.9 [A] resolution, achieving orders-of-magnitude lower background scattering compared to a liquid jet-based method. Structural comparison with the liquid jet-based model revealed similar overall structure, suggesting that the native structure is largely preserved despite dehydration during aerosolisation. Our method enables efficient SFX studies, particularly pump-probe time-resolved SFX on protein nanocrystals with enhanced signal-to-noise ratios, as well as high-throughput small molecule SFX (smSFX) applications for pharmaceuticals and functional materials.

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.

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.

We demonstrate the use of fluorescence correlation spectroscopy (FCS) to characterize fluorescently-labeled protein production. We use cell-free protein synthesis to express the protein YFP-CopB, a fusion of Chlamydia Outer Protein (Cop) B and Yellow Fluorescent Protein (YFP). CopB is a [~]50 kDa protein believed to have a critical role in chlamydial infection.1 After adding a plasmid encoding YFP-CopB to an E. coli cell-free lysate, protein expression begins. We track the cell-free reaction over several hours using the EI-FLEX, a commercial instrument with FCS capability. As protein is expressed over time, YFP-CopB increases in concentration, and the EI-FLEX detects an increase in fluorescent signal above the background of the cell-free lysate. The FCS data collected gives information about the size, aggregation tendencies, rates of production and fluorescent protein maturation, and concentration of the YFP-CopB produced. The use of FCS concurrent with cell-free synthesis presents a simple method to characterize proteins of interest as they are produced without the need for purification.

Macromolecular crystallography is limited by the phase problem: diffraction experiments measure amplitudes but not the phases required to reconstruct electron density. Existing phasing routes usually seek enough continuous phase information for density modification and model building to converge. Here, we ask how much phase information can be discarded while preserving convergence. We analyzed 14,148 diffraction datasets from chiral crystals to characterize centric reflections in reciprocal-space asymmetric units. After conditioning by centric trace and, where required, index parity, the two theoretical symmetry-allowed phase values were populated near equally, close to 50:50, independent of space group, defining a compact symmetry scaffold. We then retained this exact scaffold while compressing reference acentric phases to a one-bit alphabet {0, {pi}}; as expected from their diffuse parent distribution, the assignments were also near-balanced. Although this binary representation, with fixed attenuation 2/{pi}, introduces large angular errors (mean of 52{degrees}), it frequently supported automated structure solution: in paired Phenix AutoBuild tests, 705 of 894 binary initializers met a conservative joint criterion of final Free R [≤] 30% and relative chain recovery [≥] 70%, within a 20.0-2.5 [A] resolution window. To rank candidate seeds without rebuilding, we developed a branch-balanced Basin Score from inexpensive density-modification and map-connectivity observables computed at 20.0-3.5 [A]. The empirical score quickly separates productive from unproductive initializers before AutoBuild. Controlled phase inversion shows that basin compatibility decays gradually and can reappear in an anti-phase-related branch, indicating that buildability is not confined to a single neighborhood around the reference phase set but extends to a much broader field. These results recast phase initialization as basin entry and support future symmetry-aware, binary phase-search strategies.

In situ structural analysis allows direct visualization of protein structures in their native cellular environments, but near-atomic resolution in cellular lamellae has been significantly limited to exceptionally large complexes such as ribosomes. A key factor underlying this limitation is the degradation of data quality of thin lamellae caused by substantial subsurface damage from cryo-focused ion beam (cryo-FIB). Here, we developed a cryogenic low-energy polishing in FIB approach, which reliably produces thin lamellae with low damage across different cell types. This advance, combined with in situ single particles analysis, has pushed down the molecular weight lower limit for in situ reconstruction at near-atomic resolution to [~]400 kDa. We demonstrate this by resolving photosynthetic complexes (3.4 [A] and 3.3 [A]), metabolic enzymes (3.3 [A]), chloroplast ribosome (4.0 [A]) and respiratory chain complexes (3.7 [A]) from Chlamydomonas reinhardtii. Furthermore, the efficient workflow enables rapid structural feedback upon changes in cellular states, offering a practical way to perform multi-condition in situ structural analysis.

Light scattering in scintillators is a pervasive problem and a key factor limiting X-ray imaging resolution. Here, we shift scintillator radioluminescence from the traditional visible range into the short-wave infrared (SWIR) or near-infrared II (NIR-II, 1000-3000 nm) window to mitigate light scattering and thereby enhance light penetration and X-ray imaging resolution. We present an NIR II MgGa2O4:Ni2+ scintillator with peak emission at 1340 nm, achieving a threefold improvement in X-ray imaging resolution compared with visible scintillators owing to reduced light scattering. This heavy-metal-free NIR-II scintillator exhibits intense radioluminescence comparable to that of conventional visible-emitting CsI:Tl, achieving a detection limit of 56 nanograys per second, ~100-fold lower than typical doses used in medical imaging. We show that this NIR-II scintillator enables high-resolution X-ray radiography of electronic circuit boards and biological tissues.

The laser phase plate (LPP) enables phase-contrast imaging in cryogenic electron microscopy (cryoEM), enhancing image contrast without compromising high-resolution information. Here we report the implementation of a crossed laser phase plate (xLPP) comprising two optical cavities oriented orthogonally, installed in a ThermoFisher Scientific Krios G4 microscope equipped with a newly designed transfer lens module. We demonstrate the expected, strong contrast enhancement and stable, additive phase shifts of 90{degrees}, with a contrast transfer function (CTF) that closely matches theory. Single-particle analysis (SPA) of apoferritin, a standard benchmark sample, reached a resolution of 1.79 [A], demonstrating the system is capable of acquiring high-resolution cryoEM data. When imaging thick E. coli cells ([~]350 nm), the xLPP enhances contrast and increases low-frequency template-matching signal. Together, these results establish the feasibility of the xLPP and highlight its potential for high-contrast, high-resolution cryoEM imaging of biological systems.

Cryogenic-electron microscopy (cryo-EM) has revolutionized the field of protein structural biology. The structures of large membrane proteins are now routinely determined by cryo-EM to near atomic resolution. However, in the medium resolution range of cryo-EM maps (>[~]2 [A]), negatively charged sidechains of acidic residues are not well-resolved due to the negative electrostatic potential of the region. This may lead to incorrect sidechain models for residues like glutamic acid or aspartic acid that are central for proton transfer activity in various respiratory and photosynthetic enzymes. We previously proposed that the acidic residues with weak or non-existent cryo-EM density can be modeled to represent their low proton affinity conformations. Here, we tested this hypothesis on a larger data set of acidic amino acid residues in two high-resolution respiratory complex I structures. By using faster sidechain modeling and proton affinity prediction tools, we created a workflow that generates sidechain conformations of selected amino acid residues. We validated the sidechain conformation predictions by Q-score analysis and atomistic molecular dynamics simulations in different charged states. The proposed workflow provides a way to rapidly obtain sidechain conformations of acidic residues with weak cryo-EM densities and can be integrated into the existing cryo-EM modeling pipelines to speed up sidechain rotamer prediction.