Multi-crystal native-SAD phasing at 5 keV with a helium environment.

structure determination from single-wavelength anomalous diffraction using native sulfur or phospho-rus in biomolecules (native-SAD) is an appealing method to mitigate the labor-intensive production of heavy-atom derivatives and seleno-methio-nyl substitutions. The native-SAD method is particularly attractive for membrane proteins, which are difficult to produce and often recalcitrant to grow into decent-sized crystals. Native-SAD uses lower-energy X-rays to enhance anomalous signals from sulfur or phospho-rus.

AMX - the highly automated macromolecular crystallography (17-ID-1) beamline at the NSLS-II.

The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 10 photons s), micro-focus (7 µm × 5 µm), low-divergence (1 mrad × 0.35 mrad) energy-tunable (5-18 keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline.

The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (M).

The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or M, is a promising target for the development of novel antiviral therapeutics. Previous X-ray crystal structures of M were obtained at cryogenic tem-per-ature or room tem-per-ature only.

Hepatitis C virus NS3/4A inhibitors and other drug-like compounds as covalent binders of SARS-CoV-2 main protease.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (M also known as 3CL) is a primary drug target, due to its essential role in maturation of the viral polyproteins.

Serial crystallography with multi-stage merging of thousands of images.

KAMO and BLEND provide particularly effective tools to automatically manage the merging of large numbers of data sets from serial crystallography. The requirement for manual intervention in the process can be reduced by extending BLEND to support additional clustering options such as the use of more accurate cell distance metrics and the use of reflection-intensity correlation coefficients to infer `distances' among sets of reflections. This increases the sensitivity to differences in unit-cell parameters and allows clustering to assemble nearly complete data sets on the basis of intensity or amplitude differences.

Robotic sample changers for macromolecular X-ray crystallography and biological small-angle X-ray scattering at the National Synchrotron Light Source II. Corrigendum.

A correction in the paper by Lazo et al. [(2021). J.

Robotic sample changers for macromolecular X-ray crystallography and biological small-angle X-ray scattering at the National Synchrotron Light Source II.

Here we present two robotic sample changers integrated into the experimental stations for the macromolecular crystallography (MX) beamlines AMX and FMX, and the biological small-angle scattering (bioSAXS) beamline LiX. They enable fully automated unattended data collection and remote access to the beamlines. The system designs incorporate high-throughput, versatility, high-capacity, resource sharing and robustness.

FMX - the Frontier Microfocusing Macromolecular Crystallography Beamline at the National Synchrotron Light Source II.

Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J.

Structure of the dihydrolipoamide succinyltransferase catalytic domain from Escherichia coli in a novel crystal form: a tale of a common protein crystallization contaminant.

The crystallization of amidase, the ultimate enzyme in the Trp-dependent auxin-biosynthesis pathway, from Arabidopsis thaliana was attempted using protein samples with at least 95% purity. Cube-shaped crystals that were assumed to be amidase crystals that belonged to space group I4 (unit-cell parameters a = b = 128.6, c = 249.

Synchrotron microcrystal native-SAD phasing at a low energy.

structural evaluation of native biomolecules from single-wavelength anomalous diffraction (SAD) is a challenge because of the weakness of the anomalous scattering. The anomalous scattering from relevant native elements - primarily sulfur in proteins and phospho-rus in nucleic acids - increases as the X-ray energy decreases toward their -edge transitions. Thus, measurements at a lowered X-ray energy are promising for making native SAD routine and robust.

Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II.

Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II-AMX and FMX-deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals.

High-speed raster-scanning synchrotron serial microcrystallography with a high-precision piezo-scanner.

The Frontier Microfocus Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II with its 1 µm beam size and photon flux of 3 × 10 photons s at a photon energy of 12.66 keV has reached unprecedented dose rates for a structural biology beamline. The high dose rate presents a great advantage for serial microcrystallography in cutting measurement time from hours to minutes.

Sample manipulation and data assembly for robust microcrystal synchrotron crystallography.

With the recent developments in microcrystal handling, synchrotron microdiffraction beamline instrumentation and data analysis, microcrystal crystallo-graphy with crystal sizes of less than 10 µm is appealing at synchrotrons. However, challenges remain in sample manipulation and data assembly for robust microcrystal synchrotron crystallography. Here, the development of micro-sized polyimide well-mounts for the manipulation of microcrystals of a few micrometres in size and the implementation of a robust data-analysis method for the assembly of rotational microdiffraction data sets from many microcrystals are described.

PRIGo: a new multi-axis goniometer for macromolecular crystallography.

The Parallel Robotics Inspired Goniometer (PRIGo) is a novel compact and high-precision goniometer providing an alternative to (mini-)kappa, traditional three-circle goniometers and Eulerian cradles used for sample reorientation in macromolecular crystallography. Based on a combination of serial and parallel kinematics, PRIGo emulates an arc. It is mounted on an air-bearing stage for rotation around ω and consists of four linear positioners working synchronously to achieve x, y, z translations and χ rotation (0-90°), followed by a ϕ stage (0-360°) for rotation around the sample holder axis.

Challenges and solutions for the analysis of in situ, in crystallo micro-spectrophotometric data.

Combining macromolecular crystallography with in crystallo micro-spectrophotometry yields valuable complementary information on the sample, including the redox states of metal cofactors, the identification of bound ligands and the onset and strength of undesired photochemistry, also known as radiation damage. However, the analysis and processing of the resulting data differs significantly from the approaches used for solution spectrophotometric data. The varying size and shape of the sample, together with the suboptimal sample environment, the lack of proper reference signals and the general influence of the X-ray beam on the sample have to be considered and carefully corrected for.

Fingerprinting redox and ligand states in haemprotein crystal structures using resonance Raman spectroscopy.

It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single-crystal spectroscopies, particularly when applied in situ at macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single-crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochrome c' from Alcaligenes xylosoxidans.

D3, the new diffractometer for the macromolecular crystallography beamlines of the Swiss Light Source.

A new diffractometer for microcrystallography has been developed for the three macromolecular crystallography beamlines of the Swiss Light Source. Building upon and critically extending previous developments realised for the high-resolution endstations of the two undulator beamlines X06SA and X10SA, as well as the super-bend dipole beamline X06DA, the new diffractometer was designed to the following core design goals. (i) Redesign of the goniometer to a sub-micrometer peak-to-peak cylinder of confusion for the horizontal single axis.

Human cellular retinaldehyde-binding protein has secondary thermal 9-cis-retinal isomerase activity.

Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/vis and (1)H NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal.

A new on-axis micro-spectrophotometer for combining Raman, fluorescence and UV/Vis absorption spectroscopy with macromolecular crystallography at the Swiss Light Source.

The combination of X-ray diffraction experiments with optical methods such as Raman, UV/Vis absorption and fluorescence spectroscopy greatly enhances and complements the specificity of the obtained information. The upgraded version of the in situ on-axis micro-spectrophotometer, MS2, at the macromolecular crystallography beamline X10SA of the Swiss Light Source is presented. The instrument newly supports Raman and resonance Raman spectroscopy, in addition to the previously available UV/Vis absorption and fluorescence modes.

Selective X-ray-induced NO photodissociation in haemoglobin crystals: evidence from a Raman-assisted crystallographic study.

Despite their high physiological relevance, haemoglobin crystal structures with NO bound to haem constitute less than 1% of the total ligated haemoglobins (Hbs) deposited in the Protein Data Bank. The major difficulty in obtaining NO-ligated Hbs is most likely to be related to the oxidative denitrosylation caused by the high reactivity of the nitrosylated species with O(2). Here, using Raman-assisted X-ray crystallography, it is shown that under X-ray exposure (at four different radiation doses) crystals of nitrosylated haemoglobin from Trematomus bernacchii undergo a transition, mainly in the β chains, that generates a pentacoordinate species owing to photodissociation of the Fe-NO bond.

Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin.

Three macromolecular crystallography (MX) beamlines at the Helmholtz-Zentrum Berlin (HZB) are available for the regional, national and international structural biology user community. The state-of-the-art synchrotron beamlines for MX BL14.1, BL14.

Crystallography on a chip.

A new chip-based crystal-mounting approach for rapid room-temperature data collection from numerous crystals is described. This work was motivated by the recent development of X-ray free-electron lasers. These novel sources deliver very intense femtosecond X-ray pulses that promise to yield high-resolution diffraction data of nanocrystals before their destruction by radiation damage.