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Abstract The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein’s crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

Abstract Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses.

The JUNGFRAU detector is a well-established hybrid pixel detector developed at the Paul Scherrer Institut (PSI) designed for free-electron laser (FEL) applications. JUNGFRAU features a charge-integrating dynamic gain switching architecture, with three different gain stages and 75 μm pixel pitch. It is widely used at the European X-ray Free-Electron Laser (EuXFEL), a facility which produces high brilliance X-ray pulses at MHz repetition rate in the form of bursts repeating at 10 Hz. In nominal configuration, the detector utilizes only a single memory cell and supports data acquisition up to 2 kHz. This constrains the operation of the detector to a 10 Hz frame rate when combined with the pulsed train structure of the EuXFEL. When configured in so-called burst mode, the JUNGFRAU detector can acquire a series of images into sixteen memory cells at a maximum rate of around 150 kHz. This acquisition scheme is better suited for the time structure of the X-rays as well as the pump laser pulses at the EuXFEL. To ensure confidence in the use of the burst mode at EuXFEL, a wide range of measurements have been performed to characterize the detector, especially to validate the detector alibration procedures. In particular, by analyzing the detector response to varying photon intensity (so called ‘intensity scan’), special attention was given to the characterization of the transitions between gain stages. The detector was operated in both dynamic gain switching and fixed gain modes. Results of these measurements indicate difficulties in the characterization of the detector dynamic gain switching response while operated in burst mode, while no major issues have been found with fixed gain operation. Based on this outcome, fixed gain operation mode with all the memory cells was used during two experiments at EuXFEL, namely in serial femtosecond protein crystallography and Kossel lines measurements. The positive outcome of these two experiments validates the good results previously obtained, and opens the possibility for a wider usage of the detector in burst operation mode, although compromises are needed on the dynamic range.

Pump–probe experiments at X-ray free-electron laser (XFEL) facilities are a powerful tool for studying dynamics at ultrafast and longer timescales. Observing the dynamics in diverse scientific cases requires optical laser systems with a wide range of wavelength, flexible pulse sequences and different pulse durations, especially in the pump source. Here, the pump–probe instrumentation available for measurements at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the European XFEL is reported. The temporal and spatial stability of this instrumentation is also presented.

Characterizing the properties of X-ray free-electron laser (XFEL) sources is a critical step for optimization of performance and experiment planning. The recent availability of MHz XFELs has opened up a range of new opportunities for novel experiments but also highlighted the need for systematic measurements of the source properties. Here, MHz-enabled beam imaging diagnostics developed for the SPB/SFX instrument at the European XFEL are exploited to measure the shot-to-shot intensity statistics of X-ray pulses. The ability to record pulse-integrated two-dimensional transverse intensity measurements at multiple planes along an XFEL beamline at MHz rates yields an improved understanding of the shot-to-shot photon beam intensity variations. These variations can play a critical role, for example, in determining the outcome of single-particle imaging experiments and other experiments that are sensitive to the transverse profile of the incident beam. It is observed that shot-to-shot variations in the statistical properties of a recorded ensemble of radiant intensity distributions are sensitive to changes in electron beam current density. These changes typically occur during pulse-distribution to the instrument and are currently not accounted for by the existing suite of imaging diagnostics. Modulations of the electron beam orbit in the accelerator are observed to induce a time-dependence in the statistics of individual pulses – this is demonstrated by applying radio-frequency trajectory tilts to electron bunch-trains delivered to the instrument. We discuss how these modifications of the beam trajectory might be used to modify the statistical properties of the source and potential future applications.

An environmentally safe means of mosquito control is the application of Bacillus thuringiensis israelensis, which produces a cocktail of four naturally crystalline proteins exclusively toxic to mosquito. Here the authors report the atomic-resolution structures of Bti Cry11Aa and related Btj Cry11Ba solved de novo through Serial Femtosecond Crystallography on naturally-occurring nanocrystals.

Measurement(s) lysozyme measurement Technology Type(s) X-ray crystallography

The Sample Environment and Characterization (SEC) group of the European X-ray Free-Electron Laser (EuXFEL) develops sample delivery systems for the various scientific instruments, including systems for the injection of liquid samples that enable serial femtosecond X-ray crystallography (SFX) and single-particle imaging (SPI) experiments, among others. For rapid prototyping of various device types and materials, sub-micrometre precision 3D printers are used to address the specific experimental conditions of SFX and SPI by providing a large number of devices with reliable performance. This work presents the current pool of 3D printed liquid sample delivery devices, based on the two-photon polymerization (2PP) technique. These devices encompass gas dynamic virtual nozzles (GDVNs), mixing-GDVNs, high-viscosity extruders (HVEs) and electrospray conical capillary tips (CCTs) with highly reproducible geometric features that are suitable for time-resolved SFX and SPI experiments at XFEL facilities. Liquid sample injection setups and infrastructure on the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument are described, this being the instrument which is designated for biological structure determination at the EuXFEL.

X-ray free electron lasers deliver photon pulses that are bright enough to observe diffraction from extremely small crystals at a time scale that outruns their destruction. As crystals are continuously replaced, this technique is termed serial femtosecond crystallography (SFX). Due to its high pulse repetition rate, the European XFEL enables the collection of rich and extensive data sets, which are suited to study various scientific problems, including ultra-fast processes. The enormous data rate, data complexity, and the nature of the pixelized multimodular area detectors at the European XFEL pose severe challenges to users. To streamline the analysis of the SFX data, we developed the semiautomated pipeline EXtra-Xwiz around the established CrystFEL program suite, thereby processing diffraction patterns on detector frames into structure factors. Here we present EXtra-Xwiz, and we introduce its architecture and use by means of a tutorial. Future plans for its development and expansion are also discussed.

CCA-adding enzymes are highly specific RNA polymerases that add and maintain the sequence C-C-A at tRNA 3‘-ends. Recently, we could reveal that cold adaptation of such a polymerase is not only achieved at the expense of enzyme stability, but also at the cost of polymerization fidelity. Enzymes from psychrophilic organisms usually show an increased structural flexibility to enable catalysis at low temperatures. Here, polymerases face a dilemma, as there is a discrepancy between the need for a tightly controlled flexibility during polymerization and an increased flexibility as strategy for cold adaptation. Based on structural and biochemical analyses, we contribute to clarify the cold adaptation strategy of the psychrophilic CCA-adding enzyme from Planococcus halocryophilus, a gram-positive bacterium thriving in the arctic permafrost at low temperatures down to −15 °C. A comparison with the closely related enzyme from the thermophilic bacterium Geobacillus stearothermophilus reveals several features of cold adaptation - a significantly reduced amount of alpha-helical elements in the C-terminal tRNA-binding region and a structural adaptation in one of the highly conserved catalytic core motifs located in the N-terminal catalytic core of the enzyme.

Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine `non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.

This perspective review describes emerging techniques and future opportunities for time-resolved serial femtosecond crystallography (TR-SFX) experiments using high repetition rate XFEL sources. High repetition rate sources are becoming more available with the European XFEL in operation and the recently upgraded LCLS-II will be available in the near future. One efficient use of these facilities for TR-SFX relies on pump–probe experiments using a laser to trigger a reaction of light-responsive proteins or mix-and-inject experiments for light-unresponsive proteins. With the view to widen the application of TR-SFX, the promising field of photocaged compounds is under development, which allows the very fast laser triggering of reactions that is no longer limited to naturally light-responsive samples. In addition to reaction triggering, a key concern when performing an SFX experiment is efficient sample usage, which is a main focus of new high repetition rate-compatible sample delivery methods.

The reproducible preparation of well-diffracting crystals is a prerequisite for every structural study based on crystallography. An instrument called XtalController has recently been designed that allows the monitoring of crystallization assays using dynamic light scattering and microscopy, and integrates piezo pumps to alter the composition of the mother liquor during the experiment. We have applied this technology to study the crystallization of two enzymes, the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus, and the lysozyme from hen egg white in the presence of a synthetic chemical nucleant. We were able to (i) detect early nucleation events and (ii) drive the crystallization system (through cycles of dissolution/crystallization) toward growth conditions yielding crystals with excellent diffraction properties. This technology opens a way to the rational production of samples for crystallography, ranging from nanocrystals for electron diffraction, microcrystals for serial or conventional X-ray diffraction, to larger crystals for neutron diffraction.