Massive Compression for High Data Rate Macromolecular Crystallography (HDRMX): Impact on Diffraction Data and Subsequent Structural Analysis.

New higher-count-rate, integrating, large area X-ray detectors with framing rates as high as 17,400 images per second are beginning to be available. These will soon be used for specialized MX experiments but will require optimal lossy compression algorithms to enable systems to keep up with data throughput. Some information may be lost. Can we minimize this loss with acceptable impact on structural information? To explore this question, we have considered several approaches: summing short sequences of images, binning to create the effect of larger pixels, use of JPEG-2000 lossy wavelet-based compression, and use of Hcompress, which is a Haar-wavelet-based lossy compression borrowed from astronomy. We also explore the effect of the combination of summing, binning, and Hcompress or JPEG-2000. In each of these last two methods one can specify approximately how much one wants the result to be compressed from the starting file size. These provide particularly effective lossy compressions that retain essential information for structure solution from Bragg reflections. Synopsis: New higher-count-rate, integrating, large area X-ray detectors with framing rates as high as 17,400 images per second are beginning to be available. These will soon be used for specialized MX experiments but will require optimal lossy compression algorithms to enable systems to keep up with data throughput. Some information may be lost. Can we minimize this loss with acceptable impact on structural information?

Bragg Spot Finder (BSF): a new machine-learning-aided approach to deal with spot finding for rapidly filtering diffraction pattern images.

Macromolecular crystallography contributes significantly to understanding diseases and, more importantly, how to treat them by providing atomic resolution 3D structures of proteins. This is achieved by collecting X-ray diffraction images of protein crystals from important biological pathways. Spotfinders are used to detect the presence of crystals with usable data, and the spots from such crystals are the primary data used to solve the relevant structures. Having fast and accurate spot finding is essential, but recent advances in synchrotron beamlines used to generate X-ray diffraction images have brought us to the limits of what the best existing spotfinders can do. This bottleneck must be removed so spotfinder software can keep pace with the X-ray beamline hardware improvements and be able to see the weak or diffuse spots required to solve the most challenging problems encountered when working with diffraction images. In this paper, we first present Bragg Spot Detection (BSD), a large benchmark Bragg spot image dataset that contains 304 images with more than 66 000 spots. We then discuss the open source extensible U-Net-based spotfinder Bragg Spot Finder (BSF), with image pre-processing, a U-Net segmentation backbone, and post-processing that includes artifact removal and watershed segmentation. Finally, we perform experiments on the BSD benchmark and obtain results that are (in terms of accuracy) comparable to or better than those obtained with two popular spotfinder software packages (Dozor and DIALS), demonstrating that this is an appropriate framework to support future extensions and improvements.

Investigation of fast and efficient lossless compression algorithms for macromolecular crystallography experiments.

Structural biology experiments benefit significantly from state-of-the-art synchrotron data collection. One can acquire macromolecular crystallography (MX) diffraction data on large-area photon-counting pixel-array detectors at framing rates exceeding 1000 frames per second, using 200 Gbps network connectivity, or higher when available. In extreme cases this represents a raw data throughput of about 25 GB s-1, which is nearly impossible to deliver at reasonable cost without compression. Our field has used lossless compression for decades to make such data collection manageable. Many MX beamlines are now fitted with DECTRIS Eiger detectors, all of which are delivered with optimized compression algorithms by default, and they perform well with current framing rates and typical diffraction data. However, better lossless compression algorithms have been developed and are now available to the research community. Here one of the latest and most promising lossless compression algorithms is investigated on a variety of diffraction data like those routinely acquired at state-of-the-art MX beamlines.

EstG is a novel esterase required for cell envelope integrity in Caulobacter.

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the ⍺-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

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 × 1012 photons s-1), 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. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented.

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. If the data sets are already sufficiently complete to permit it, one applies KAMO once and clusters the data using intensities only. When starting from incomplete data sets, one applies KAMO twice, first using unit-cell parameters. In this step, either the simple cell vector distance of the original BLEND or the more sensitive NCDist is used. This step tends to find clusters of sufficient size such that, when merged, each cluster is sufficiently complete to allow reflection intensities or amplitudes to be compared. One then uses KAMO again using the correlation between reflections with a common hkl to merge clusters in a way that is sensitive to structural differences that may not have perturbed the unit-cell parameters sufficiently to make meaningful clusters. Many groups have developed effective clustering algorithms that use a measurable physical parameter from each diffraction still or wedge to cluster the data into categories which then can be merged, one hopes, to yield the electron density from a single protein form. Since these physical parameters are often largely independent of one another, it should be possible to greatly improve the efficacy of data-clustering software by using a multi-stage partitioning strategy. Here, one possible approach to multi-stage data clustering is demonstrated. The strategy is to use unit-cell clustering until the merged data are sufficiently complete and then to use intensity-based clustering. Using this strategy, it is demonstrated that it is possible to accurately cluster data sets from crystals that have subtle differences.

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 (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials.

A simple technique to classify diffraction data from dynamic proteins according to individual polymorphs.

One often observes small but measurable differences in the diffraction data measured from different crystals of a single protein. These differences might reflect structural differences in the protein and may reveal the natural dynamism of the molecule in solution. Partitioning these mixed-state data into single-state clusters is a critical step that could extract information about the dynamic behavior of proteins from hundreds or thousands of single-crystal data sets. Mixed-state data can be obtained deliberately (through intentional perturbation) or inadvertently (while attempting to measure highly redundant single-crystal data). To the extent that different states adopt different molecular structures, one expects to observe differences in the crystals; each of the polystates will create a polymorph of the crystals. After mixed-state diffraction data have been measured, deliberately or inadvertently, the challenge is to sort the data into clusters that may represent relevant biological polystates. Here, this problem is addressed using a simple multi-factor clustering approach that classifies each data set using independent observables, thereby assigning each data set to the correct location in conformational space. This procedure is illustrated using two independent observables, unit-cell parameters and intensities, to cluster mixed-state data from chymotrypsinogen (ChTg) crystals. It is observed that the data populate an arc of the reaction trajectory as ChTg is converted into chymotrypsin.

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. Synchrotron Rad. 28, 1649-1661] is made.

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. All systems are centered around a six-axis industrial robotic arm coupled with a force torque sensor and in-house end effectors (grippers). They have the same software architecture and the facility standard EPICS-based BEAST alarm system. The MX system is compatible with SPINE bases and Unipucks. It comprises a liquid nitrogen dewar holding 384 samples (24 Unipucks) and a stay-cold gripper, and utilizes machine vision software to track the sample during operations and to calculate the final mount position on the goniometer. The bioSAXS system has an in-house engineered sample storage unit that can hold up to 360 samples (20 sample holders) which keeps samples at a user-set temperature (277 K to 300 K). The MX systems were deployed in early 2017 and the bioSAXS system in early 2019.

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. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5-30 keV photon energy range and delivers a flux of 4.0 × 1012 photons s-1 at 1 Šinto a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 1012 photons s-1 at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods - serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362-1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238-246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.

Future trends in synchrotron science at NSLS-II.

In this paper, we summarize briefly some of the future trends in synchrotron science as seen at the National Synchrotron Light Source II, a new, low emittance source recently commissioned at Brookhaven National Laboratory. We touch upon imaging techniques, the study of dynamics, the increasing use of multimodal approaches, the vital importance of data science, and other enabling technologies. Each are presently undergoing a time of rapid change, driving the field of synchrotron science forward at an ever increasing pace. It is truly an exciting time and one in which Roger Cowley, to whom this journal issue is dedicated, would surely be both invigorated by, and at the heart of.

High-resolution structure of intramolecularly proteolyzed human mucin-1 SEA domain.

SEA domains are ubiquitous in large proteins associated with highly glycosylated environments. Certain SEA domains undergo intramolecular proteolysis involving a nucleophilic attack of a serine hydroxyl group on the preceding glycine carbonyl. The mucin-1 (MUC1) SEA domain has been extensively investigated as a model of intramolecular proteolysis. Since neither a general base, a general acid, nor an oxyanion hole could be identified in MUC1 SEA, it has been suggested that proteolysis is accelerated by a non-planarity of the scissile peptide bond imposed by protein folding. A reactant distorted peptide bond has been also invoked to explain the autoproteolysis of several unrelated proteins. However, the only evidence of peptide distortion in MUC1 SEA stems from molecular dynamic simulations of the reactant modeled upon a single NMR structure of the cleaved product. We report the first high-resolution X-ray structure of cleaved MUC1 SEA. Structural comparison with uncleaved SEA domains suggests that the number of residues evolutionarily inserted in the cleaved loop of MUC1 SEA precludes the formation of a properly hydrogen-bonded beta turn. By sequence analysis, we show that this conformational frustration is shared by all known cleaved SEA domains. In addition, alternative conformations of the uncleaved precursor could be modeled in which the scissile peptide bond is planar. The implications of these structures for autoproteolysis are discussed in the light of the previous research on autoproteolysis.

Best practices for high data-rate macromolecular crystallography (HDRMX).

In macromolecular crystallography, higher flux, smaller beams, and faster detectors open the door to experiments with very large numbers of very small samples that can reveal polymorphs and dynamics but require re-engineering of approaches to the clustering of images both at synchrotrons and XFELs (X-ray free electron lasers). The need for the management of orders of magnitude more images and limitations of file systems favor a transition from simple one-file-per-image systems such as CBF to image container systems such as HDF5. This further increases the load on computers and networks and requires a re-examination of the presentation of metadata. In this paper, we discuss three important components of this problem-improved approaches to the clustering of images to better support experiments on polymorphs and dynamics, recent and upcoming changes in metadata for Eiger images, and software to rapidly validate images in the revised Eiger format.

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. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins-Akt1, PI3Kα, and CDP-Chase-to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or "raster-collect", a more automated approach for a larger number of crystals (using CDP-Chase as an example).

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into ß-lactam resistance.

The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of ß-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the ß-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three ß-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.

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 × 1012 photons s-1 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. To provide the instrumentation basis for such measurements at the full flux of the FMX beamline, a high-speed, high-precision goniometer based on a unique XYZ piezo positioner has been designed and constructed. The piezo-based goniometer is able to achieve sub-100 nm raster-scanning precision at over 10 grid-linepairs s-1 frequency for fly scans of a 200 µm-wide raster. The performance of the scanner in both laboratory and serial crystallography measurements up to the maximum frame rate of 750 Hz of the Eiger 16M's 4M region-of-interest mode has been verified in this work. This unprecedented experimental speed significantly reduces serial-crystallography data collection time at synchrotrons, allowing utilization of the full brightness of the emerging synchrotron radiation facilities.

Structural Insights into the Induced-fit Inhibition of Fascin by a Small-Molecule Inhibitor.

Tumor metastasis is responsible for ~90% of all cancer deaths. One of the key steps of tumor metastasis is tumor cell migration and invasion. Filopodia are cell surface extensions that are critical for tumor cell migration. Fascin protein is the main actin-bundling protein in filopodia. Small-molecule fascin inhibitors block tumor cell migration, invasion, and metastasis. Here we present the structural basis for the mechanism of action of these small-molecule fascin inhibitors. X-ray crystal structural analysis of a complex of fascin and a fascin inhibitor shows that binding of the fascin inhibitor to the hydrophobic cleft between the domains 1 and 2 of fascin induces a ~35o rotation of domain 1, leading to the distortion of both the actin-binding sites 1 and 2 on fascin. Furthermore, the crystal structures of an inhibitor alone indicate that the conformations of the small-molecule inhibitors are dynamic. Mutations of the inhibitor-interacting residues decrease the sensitivity of fascin to the inhibitors. Our studies provide structural insights into the molecular mechanism of fascin protein function as well as the action of small-molecule fascin inhibitors.

Loss of a Functionally and Structurally Distinct ld-Transpeptidase, LdtMt5, Compromises Cell Wall Integrity in Mycobacterium tuberculosis.

The final step of peptidoglycan (PG) biosynthesis in bacteria involves cross-linking of peptide side chains. This step in Mycobacterium tuberculosis is catalyzed by ld- and dd-transpeptidases that generate 3→3 and 4→3 transpeptide linkages, respectively. M. tuberculosis PG is predominantly 3→3 cross-linked, and LdtMt2 is the dominant ld-transpeptidase. There are four additional sequence paralogs of LdtMt2 encoded by the genome of this pathogen, and the reason for this apparent redundancy is unknown. Here, we studied one of the paralogs, LdtMt5, and found it to be structurally and functionally distinct. The structures of apo-LdtMt5 and its meropenem adduct presented here demonstrate that, despite overall architectural similarity to LdtMt2, the LdtMt5 active site has marked differences. The presence of a structurally divergent catalytic site and a proline-rich C-terminal subdomain suggest that this protein may have a distinct role in PG metabolism, perhaps involving other cell wall-anchored proteins. Furthermore, M. tuberculosis lacking a functional copy of LdtMt5 displayed aberrant growth and was more susceptible to killing by crystal violet, osmotic shock, and select carbapenem antibiotics. Therefore, we conclude that LdtMt5 is not a functionally redundant ld-transpeptidase, but rather it serves a unique and important role in maintaining the integrity of the M. tuberculosis cell wall.

Crystallization of lysozyme with (R)-, (S)- and (RS)-2-methyl-2,4-pentanediol.

Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.