Serial femtosecond crystallography (SFX) and continuous serial electron diffraction (c-SerialED) both enable high-resolution structure determination from protein microcrystals with minimal radiation damage, making it ideal for studying redox-active metalloenzymes. Here, c-SerialED and SFX were used to solve structures of the class Ia ribonucleotide reductase R2 subunit in oxidized (FeIII-FeIII), reduced (FeII-FeII), and re-oxidized states at ∼1.8 Å resolution, capturing three points in a redox reaction. These results demonstrate that c-SerialED can track reversible changes at the redox-site, enabling future time-resolved studies. Comparison between c-SerialED structures and SFX diffraction and emission data confirmed minimal radiation damage. Furthermore, previously reported structures use mercury in the crystallization condition and show mercury-induced conformational changes. Here, we use mercury-free crystallization conditions and reveal a water molecule in the redox center of the reduced state, absent in the previous structures, making these structures more representative of the physiological state.

During photosynthetic water oxidation, the Mn4Ca cluster in Photosystem II progresses through five intermediate Si (i = 0-4) states. X-ray crystallography studies have reported the insertion of one new O ligand during the formation of the S3 state, but recent studies question the presence of this additional ligand based on cryo-EM and earlier room-temperature crystallography data. There is also controversy about whether the O-O bond interaction already occurs in the S3 state or in the subsequent S3 to S0 transition. Here we report conventional high-resolution data for the S1, S2, and S3 states to a resolution of ~1.9 Å, and anomalous diffraction data at two energies (9.5 keV and 7 keV), that was used to model the Mn positions, followed by determination of oxygen positions using the high-resolution maps. We show that the new oxygen atom, OX (or O6), in the S3 state is observable as a distinct peak without any restraints, confirming its ligation to Mn1 and Ca. The OX-O5 distance is ~2.1 Å, supporting no strong interaction between them in the S3 state, suggesting that if this is the O-O bond formation site, it is formed during the S3 to S0 transition initiated by the final oxidation of the cluster.

X-ray crystallography is increasingly employed to study protein conformational ensembles under physiological conditions, but the effects of the crystal lattice on protein motions remains understudied. Here, we report the structure determination of soybean lipoxygenase-1 (SLO) from microcrystal slurries using serial femtosecond crystallography (SFX) at the Linac Coherent Light Source. During data analysis, we observed unexpected polymorphism in SLO 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 crystal populations consistent with different solvent content. By combining unit-cell clustering with systematic reindexing, we resolved two distinct polymorphs and determined two independent structures from a single experiment. The two structures exhibit a small overall RMSD (0.34 Å), yet a difference distance matrix reveals coordinated rearrangements that are not readily apparent from simple structural overlays. Furthermore, a difference of approximately 8.5% in crystal solvent content produces measurable differences in crystal contacts and conformational flexibility. The more hydrated (large-cell) polymorph exhibits greater inter-domain flexibility, as well as higher B-factors in key hydrophobic core residues. In the dehydrated (small-cell) polymorph, which shows less interdomain flexibility, these same residues adopt discrete alternative conformations resolvable in the electron density. Our results highlight that subtle changes in crystal packing can give rise to distinct conformational landscapes for crystallized proteins, with potential implications for the interpretation of protein intramolecular dynamics from crystallographic data.

Metal-organic chalcogenolates (MOChas) are hybrid materials composed of metal-chalcogenide networks coordinated by organic ligands, offering a versatile platform for structural and electronic tunability. The use of molecular ligand design to steer material formation represents a powerful strategy for accessing new solid-state topologies. In this work, we report two new silver benzenethiolate MOChas incorporating protic meta-functionalized ligands─hydroxy (-OH) and amine (-NH2)─which exhibit hydrogen-bond-driven supramolecular organization and novel inorganic connectivities. Rather than modifying existing materials, we contextualize these compounds as distinct outcomes within a structural continuum. Silver para- and meta-methoxy-benzenethiolates (p-OCH3 and m-OCH3) serve as control points for known 2D and 1D topologies, respectively. The new materials, m-OH and m-NH2, were structurally characterized using small molecule serial femtosecond crystallography (smSFX), and their intermediate energetic and electronic properties were confirmed through density functional theory (DFT) calculations. We introduce the concept of supramolecular distortion to describe how ligand-driven intermolecular interactions reshape inorganic topology─not as deviations from a fixed state, but as distinct, kinetically accessible ground-state architectures. This work establishes a design paradigm linking organic ligand identity to predictable shifts in inorganic dimensionality in MOChas.

In all photosynthetic reaction centers (RC), transmembrane electron-transfer (ET) cofactor pathways are coordinated by homologous peptides and arranged with approximate C2 symmetry, providing two possible paths for charge separation (designated A and B). In type II RCs-Photosystem II and the purple bacterial RCs-only the A branch is active for ET. A variant bacterial RC containing nine amino acid substitutions that result in high-yield ET along the normally nonfunctional B branch was designed. Structural and theoretical studies were combined to understand factors that control unique ET reactions in the complex. Serial femtosecond crystallography performed at an X-ray Free Electron Laser afforded the room temperature X-ray-damage-free crystal structure of the variant RC, revealing interactions between the substituted amino acids and the ET cofactors. The positions of the bacteriochlorin cofactors on the symmetry-related pathways are unaffected, underscoring that the substitutions alter the energetics of ET, leading to the functional changes. In the binding pockets for the terminal electron acceptor quinones, alterations in the positions of neighboring amino acids and water molecules are coordinated with the change of the substituted side chains. Modifications deactivated A-branch ET and activated transmembrane charge separation along the B branch. Electrostatic calculations based on the structure reveal how differences in stabilization of charge-separated states by the protein environment underlie the change in direction of electron flow in this variant RC and provide insights into mechanisms by which B-branch charge separation across the photosynthetic membrane can be achieved in high yield.

This paper is a report of the High Data Rate Macromolecular Crystallography workshop held on 23 July 2025 as part of the 2025 meeting of the American Crystallographic Association in Lombard, IL, USA, 18-23 July 2025. This report summarizes the discussions, questions, action items, and recommendations that arose from the meeting and includes links to the presentations. The sessions were moderated by Aaron S. Brewster and Graeme Winter. There was particularly lively discussion about the possible need for lossy compression as data rates increase, as multimodal experiments become more popular and as research budgets are squeezed.

Our work reveals the structure of the active state of Methyl-Coenzyme M Reductase (MCR), the key and rate-limiting enzyme in biological methane formation. We find large differences between the active Ni(I) and inactive Ni(II) proteins and provide insight into how nature makes and breaks the C-H bond of methane. The Ni(II)-F430 center in inactive MCR contains four planar nitrogen ligands, a lower axial glutamine oxo, and an upper axial thiolate. The Ni(I)-enzyme replaces the axial ligands with a single water. The one-electron redox change results in movement of the Ni ion and upward swing of the β-lactam ring in the tetrapyrrole coupled to a domino-like protein quake through second sphere residues, inter-subunit interactions, a substrate tunnel, affecting even the dimensions of the unit cell. These structural changes lead Ni(I)-MCR to release a charge clamp that, in the Ni(II) state, locks down substrate Coenzyme B. Determining the Ni(I)-MCR structure required development of rigorous anaerobic crystallographic techniques. Validation of the MCR redox state was accomplished by in-line and parallel spectroscopic and unit cell analyses. This structure has large implications for developing technologies to limit methane emissions and efficiently produce biofuels. Methodology described here will enhance structural biology for other oxygen-sensitive enzymes. ### Competing Interest Statement The authors have declared no competing interest. Office of Basic Energy SciencesOffice of Basic Energy Sciences, https://ror.org/05mg91w61, DE-FG02-08ER15931, FWP 100593, DE-AC02-05CH11231, DE-SC0014664, DE-AC02-76SF00515, DEAC02-05CH11231 National Institutes of HealthNational Institutes of Health, https://ror.org/01cwqze88, GM149528, GM110501, GM126289, GM117126, GM151988, P30GM133894

Photosynthesis provides most of the bio-available energy and oxygen to our planet. Yet, while several other mechanisms in oxygenic and anoxygenic photosynthesis have been thoroughly investigated, the core water-splitting reaction of photosystem II (PSII) remains largely uncharacterized. The recent advent of X-ray free-electron lasers has provided us with a tool to probe the structure of PSII's oxygen-evolving complex under ambient conditions and with microsecond time resolution. Still, the quality of diffraction data offered by these experiments is insufficient to reliably observe one-electron differences between individual time points.[1] Instead of probing the imprecise scatterer distribution, oxidation states of individual metal atoms can be assigned by investigating their X-ray absorption edges. Information from classical spectroscopy can not be matched to individual atoms; however, by performing serial diffraction experiments with a pink beam tuned to the metal absorption edge, the anomalous dispersion of each atom becomes embedded in the diffraction image. A careful analysis of Bragg reflection profiles can be thus applied to retrieve the atomic form factors as a function of energy. Refined absorption curves can be then used to characterize the electronic structure of each atom. This spatially resolved anomalous dispersion (SPREAD) technique has been previously successfully applied to data simulated for ferredoxin: a 25 kDa protein containing two differently charged iron centers.[2] The present work describes our recent advances in scaling the pipeline for a 750 kDa PSII with a four-manganese cluster and adapting it to experimental data. In particular, we describe the first working refinement of experimental data, as well as issues encountered with reliability, restraints, mosaicity, and memory use.

When serial crystallography at XFELs was first being developed, it was unclear how it should best be used. Early experiments included studying single crystals, or weak or small crystals, or sample delivery methods that over used crystal material or under used the pulse rate. It took years of refinements to find the strengths of this method, including pump/probe experiments, room-temperature structure determination, damage-free chemistry/function measurements, exploration of dynamics, and more recently, small molecule serial femtosecond crystallography (smSFX). Now, interest in the field is exploding, with serial beamlines being constructed at synchrotrons, either from scratch or being added onto existing instruments. In this talk, several serial crystallography projects will be presented, including multi-modal methods, ligand screening at the ALS, and the transformative method of smSFX.

Serial crystallography is an important technique with unique abilities to resolve enzymatic transition states, minimize radiation damage to sensitive metalloenzymes and perform de novo structure determination from micrometre-sized crystals. This technique requires the merging of data from thousands of crystals, making manual identification of errant crystals unfeasible. cctbx.xfel.merge uses filtering to remove problematic data. However, this process is imperfect, and data reduction must be robust to outliers. We add robustness to cctbx.xfel.merge at the step of uncertainty determination for reflection intensities. This step is a critical point for robustness because it is the first step where the data sets are considered as a whole, as opposed to individual lattices. Robustness is conferred by reformulating the error-calibration procedure to have fewer and less stringent statistical assumptions and incorporating the ability to down-weight low-quality lattices. We then apply this method to five macromolecular XFEL data sets and observe the improvements to each. The appropriateness of the intensity uncertainties is demonstrated through internal consistency. This is performed through theoretical CC1/2 and I/σ relationships and by weighted second moments, which use Wilson's prior to connect intensity uncertainties with their expected distribution. This work presents new mathematical tools to analyze intensity statistics and demonstrates their effectiveness through the often underappreciated process of uncertainty analysis.

X-ray absorption spectroscopy (XAS) of 3d transition metals provides important electronic structure information for many fields. However, X-ray-induced radiation damage under physiological temperature has prevented using this method to study dilute aqueous systems, such as metalloenzymes, as the catalytic reaction proceeds. Here we present a new approach to enable operando XAS of dilute biological samples and demonstrate its feasibility with K-edge XAS spectra from the Mn cluster in photosystem II and the Fe-S centers in photosystem I. This approach combines highly efficient sample delivery strategies and a robust signal normalization method with high-transmission Bragg diffraction-based spectrometers at X-ray free-electron lasers (XFELs) in a damage-free, shot-by-shot mode. These photon-out spectrometers have been optimized for discriminating the metal Mn/Fe Kα fluorescence signals from the overwhelming scattering background present on currently available detectors for XFELs that lack suitable energy discrimination. We quantify the enhanced performance metrics of the spectrometer and discuss its potential applications for acquiring time-resolved XAS spectra of biological samples during their reactions at XFELs.

The photoreaction and commensurate structural changes of a chromophore within biological photoreceptors elicit conformational transitions of the protein promoting the switch between deactivated and activated states. We investigated how this coupling is achieved in a bacterial phytochrome variant, Agp2-PAiRFP2. Contrary to classical protein crystallography, which only allows probing (cryo-trapped) stable states, we have used time-resolved serial femtosecond x-ray crystallography (tr-SFX) and pump-probe techniques with various illumination and delay times with respect to photoexcitation of the parent Pfr state. Thus, structural data for seven time frames were sorted into groups of molecular events along the reaction coordinate. They range from chromophore isomerization to the formation of Meta-F, the intermediate that precedes the functional relevant secondary structure transition of the tongue. Structural data for the early events were used to calculate the photoisomerization pathway to complement the experimental data. Late events allow identifying the molecular switch that is linked to the intramolecular proton transfer as a prerequisite for the following structural transitions.

The cctbx.xfel suite of processing programs and tools allows fast, visual analysis of serial diffraction images from synchrotrons and XFELs. Built on DIALS and cctbx, cctbx.xfel is designed for real-time and post-experiment processing with a fully featured graphical user interface. Users can quickly identify hitrates, view diffraction patterns, analyze unit-cell isomorphism using clustering, and merge data using a metadata tagging approach that allows on-the-fly organization and visualization of processing results. This paper describes the fundamental algorithms and command-line programs used by cctbx.xfel, including the two main program dials.stills_process, which performs spot-finding, indexing, geometric refinement, and integration, and cctbx.xfel.merge, which performs scaling, post-refinement, and merging. A discussion of merging statistics is presented and newer features are described, including random sub-sampling for indexing multi-lattice hits and ΔCC 1/2 filtering to remove outliers. Finally we show a complex, heterogeneous sample containing hexagonal and monoclinic isoforms in P63 and P21. The isoforms are separated by unit cell clustering, and for each isoform we resolve a (pseudo-)merohedral indexing ambiguity.

In macromolecular crystallography (MX), integrated intensities’ final uncertainties are typically calculated by an empirical transformation of initial counting statistics uncertainties, a procedure generally known as error calibration. Existing MX error calibration algorithms were designed for single-crystal rotational diffraction experiments. This work describes their further adaptation to serial femtosecond crystallography and is motivated by fundamental differences in data and experimentation between these methods. We propose a new parameterized error transformation that applies different levels of error to each lattice based on its correlation to a scaling reference. A maximum likelihood approach is used to robustly optimize the transformation’s parameters. This algorithm is implemented within cctbx.xfel.merge and is demonstrated to improve the accuracy of the uncertainty estimates.In macromolecular crystallography (MX), integrated intensities’ final uncertainties are typically calculated by an empirical transformation of initial counting statistics uncertainties, a procedure generally known as error calibration. Existing MX error calibration algorithms were designed for single-crystal rotational diffraction experiments. This work describes their further adaptation to serial femtosecond crystallography and is motivated by fundamental differences in data and experimentation between these methods. We propose a new parameterized error transformation that applies different levels of error to each lattice based on its correlation to a scaling reference. A maximum likelihood approach is used to robustly optimize the transformation’s parameters. This algorithm is implemented within cctbx.xfel.merge and is demonstrated to improve the accuracy of the uncertainty estimates.

Catalysis by metalloenzymes is controlled by precise movement of protons and electrons at active sites which are often coordinated with ligand binding, side-chain movement, or larger protein conformational changes. We seek to understand the mechanism of Photosystem II (PSII), where the coordinated movement of substrate and electrons within a Mn4CaO5 cofactor catalyzes biological water oxidation. To fully characterize mechanisms of PSII and other enzymes, simultaneous interrogation of protein structure and oxidation state of each metal cofactor is required. These research questions can be addressed by X-ray crystallography by analyzing the anomalous scattering of each metal atom, even for metallocofactors which contain multiple copies of the same metal type. The spatially resolved anomalous dispersion (SPREAD) method depends on the wavelength dependent behavior of scatterers around their absorption K-edge. Time-resolved crystallographic methods using X-ray free electron lasers (XFELs) could provide spatial and temporal resolution of oxidation states, but adapting SPREAD for use with XFELs is inherently challenging due to the shot-to-shot variability and stochastic nature of the XFEL pulses. We present progress towards extending the SPREAD method to XFELs, taking advantage of the pulse distribution to collect entire datasets without the need for monochromatic X-rays, and discuss methods for data processing. With one data analysis method, energy contributions to each pixel contributing to Bragg spots are computationally modeled, allowing structure factors to be extracted. As an alternative to the pixel-based approach, we also explore assignment of energies to whole spots as a means to determine energy dependent scattering factors. Developing the software for time-resolved SPREAD experiments enables serial crystallography experiments to isolate transient intermediates that are important in catalysis, including the four metastable intermediates in the catalytic cycle of PSII, and the elusive S4 state where O-O bond formation occurs.

Most X-ray sources are inherently polychromatic. Polychromatic (“pink”) X-rays provide an efficient way to conduct diffraction experiments as many more photons can be used and large regions of reciprocal space can be probed without sample rotation during exposure—ideal conditions for time-resolved applications. Analysis of such data is complicated, however, causing most X-ray facilities to discard>99% of X-ray photons to obtain monochromatic data. Key challenges in analyzing polychromatic diffraction data include lattice searching, indexing and wavelength assignment, correction of measured intensities for wavelength-dependent effects, and deconvolution of harmonics. We recently described an algorithm, Careless, that can perform harmonic deconvolution and correct measured intensities for variation in wavelength when presented with integrated diffraction intensities and assigned wavelengths. Here, we present Laue-DIALS, an open-source software pipeline that indexes and integrates polychromatic diffraction data. Laue-DIALS is based on the dxtbx toolbox, which supports the DIALS software commonly used to process monochromatic data. As such, Laue-DIALS provides many of the same advantages: an open-source, modular, and extensible architecture, providing a robust basis for future development. We present benchmark results showing that Laue-DIALS, together with Careless, provides a suitable approach to the analysis of polychromatic diffraction data, including for time-resolved applications.

X-ray scattering experiments using Free Electron Lasers (XFELs) are a powerful tool to determine the molecular structure and function of unknown samples (such as COVID-19 viral proteins). XFEL experiments are a challenge to computing in two ways: i) due to the high cost of running XFELs, a fast turnaround time from data acquisition to data analysis is essential to make informed decisions on experimental protocols; ii) data collection rates are growing exponentially, requiring new scalable algorithms. Here we report our experiences analyzing data from two experiments at the Linac Coherent Light Source (LCLS) during September 2020. Raw data were analyzed on NERSC's Cori XC40 system, using the Superfacility paradigm: our workflow automatically moves raw data between LCLS and NERSC, where it is analyzed using the software package CCTBX. We achieved real time data analysis with a turnaround time from data acquisition to full molecular reconstruction in as little as 10 min -- sufficient time for the experiment's operators to make informed decisions. By hosting the data analysis on Cori, and by automating LCLS-NERSC interoperability, we achieved a data analysis rate which matches the data acquisition rate. Completing data analysis with 10 mins is a first for XFEL experiments and an important milestone if we are to keep up with data collection trends.

SummaryThe upcoming exascale computing systems Frontier and Aurora will draw much of their computing power from GPU accelerators. The hardware for these systems will be provided by AMD and Intel, respectively, each supporting their own GPU programming model. The challenge for applications that harness one of these exascale systems will be to avoid lock‐in and to preserve performance portability. We report here on our results of using Kokkos to accelerate a real‐world application on NERSC's Perlmutter Phase 1 (using NVIDIA A100 accelerators) and Crusher, the testbed system for OLCF's Frontier (using AMD MI250X). By porting to Kokkos, we successfully ran the same X‐ray tracing code on both systems and achieved speed‐ups between 13 % and 66 % compared to the original CUDA code. These results are a highly encouraging demonstration of using Kokkos to accelerate production science code.

ExaFEL is an HPC-capable X-ray Free Electron Laser (XFEL) data analysis software suite for both Serial Femtosecond Crystallography (SFX) and Single Particle Imaging (SPI) developed in collaboration with the Linac Coherent Lightsource (LCLS), Lawrence Berkeley National Laboratory (LBNL) and Los Alamos National Laboratory. ExaFEL supports real-time data analysis via a cross-facility workflow spanning LCLS and HPC centers such as NERSC and OLCF. Our work therefore constitutes initial path-finding for the US Department of Energy's (DOE) Integrated Research Infrastructure (IRI) program. We present the ExaFEL team's 7 years of experience in developing real-time XFEL data analysis software for the DOE's exascale supercomputers. We present our experiences and lessons learned with the Perlmutter and Frontier supercomputers. Furthermore we outline essential data center services (and the implications for institutional policy) required for real-time data analysis. Finally we summarize our software and performance engineering approaches and our experiences with NERSC's Perlmutter and OLCF's Frontier systems. This work is intended to be a practical blueprint for similar efforts in integrating exascale compute resources into other cross-facility workflows.

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn 4 CaO 5 cluster first stores four oxidizing equivalents, the S 0 to S 4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O–O bond formation chemistry 1 – 3 . Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok’s photosynthetic water oxidation cycle, the S 3 →[S 4 ]→S 0 transition where O 2 is formed and Kok’s water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn 4 CaO 5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom O x , which was introduced as a bridging ligand between Ca and Mn1 during the S 2 →S 3 transition 4 – 6 , disappears or relocates in parallel with Y z reduction starting at approximately 700 μs after the third flash. The onset of O 2 evolution, as indicated by the shortening of the Mn1–Mn4 distance, occurs at around 1,200 μs, signifying the presence of a reduced intermediate, possibly a bound peroxide.