In situ serial crystallography facilitates 96-well plate structural analysis at low symmetry.

The advent of serial crystallography has rejuvenated and popularized room-temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g. large complexes, membrane proteins and viruses) form fragile crystals that are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallization. Many medically relevant samples are expressed in mammalian cell lines, producing a meager quantity of protein that is incompatible with batch crystallization. This can limit the scope of serial crystallography approaches. Direct in situ data collection from a 96-well crystallization plate enables not only the identification of the best diffracting crystallization condition but also the possibility for structure determination under ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallization plates mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility. We applied our iSX approach on a challenging project, autotaxin, a therapeutic target expressed in a stable human cell line, to determine the structure in the lowest-symmetry P1 space group at 3.0 Šresolution. Our in situ data collection strategy provided a complete dataset for structure determination while screening various crystallization conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallization plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons.

ID23-2: an automated and high-performance microfocus beamline for macromolecular crystallography at the ESRF. Corrigendum.

A revised version of Table 2 of Nanao et al. [J. Synchrotron Rad. (2022). 29, 581-590] is provided.

ID23-2: an automated and high-performance microfocus beamline for macromolecular crystallography at the ESRF.

ID23-2 is a fixed-energy (14.2 keV) microfocus beamline at the European Synchrotron Radiation Facility (ESRF) dedicated to macromolecular crystallography. The optics and sample environment have recently been redesigned and rebuilt to take full advantage of the upgrade of the ESRF to the fourth generation Extremely Brilliant Source (ESRF-EBS). The upgraded beamline now makes use of two sets of compound refractive lenses and multilayer mirrors to obtain a highly intense (>1013 photons s-1) focused microbeam (minimum size 1.5 µm × 3 µm full width at half-maximum). The sample environment now includes a FLEX-HCD sample changer/storage system, as well as a state-of-the-art MD3Up high-precision multi-axis diffractometer. Automatic data reduction and analysis are also provided for more advanced protocols such as synchrotron serial crystallographic experiments.

A comparative anatomy of protein crystals: lessons from the automatic processing of 56†000 samples.

The fully automatic processing of crystals of macromolecules has presented a unique opportunity to gather information on the samples that is not usually recorded. This has proved invaluable in improving sample-location, characterization and data-collection algorithms. After operating for four years, MASSIF-1 has now processed over 56 000 samples, gathering information at each stage, from the volume of the crystal to the unit-cell dimensions, the space group, the quality of the data collected and the reasoning behind the decisions made in data collection. This provides an unprecedented opportunity to analyse these data together, providing a detailed landscape of macromolecular crystals, intimate details of their contents and, importantly, how the two are related. The data show that mosaic spread is unrelated to the size or shape of crystals and demonstrate experimentally that diffraction intensities scale in proportion to crystal volume and molecular weight. It is also shown that crystal volume scales inversely with molecular weight. The results set the scene for the development of X-ray crystallography in a changing environment for structural biology.

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules.

High-brilliance X-ray beams coupled with automation have led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

MXCuBE2: the dawn of MXCuBE Collaboration.

MXCuBE2 is the second-generation evolution of the MXCuBE beamline control software, initially developed and used at ESRF - the European Synchrotron. MXCuBE2 extends, in an intuitive graphical user interface (GUI), the functionalities and data collection methods available to users while keeping all previously available features and allowing for the straightforward incorporation of ongoing and future developments. MXCuBE2 introduces an extended abstraction layer that allows easy interfacing of any kind of macromolecular crystallography (MX) hardware component, whether this is a diffractometer, sample changer, detector or optical element. MXCuBE2 also works in strong synergy with the ISPyB Laboratory Information Management System, accessing the list of samples available for a particular experimental session and associating, either from instructions contained in ISPyB or from user input via the MXCuBE2 GUI, different data collection types to them. The development of MXCuBE2 forms the core of a fruitful collaboration which brings together several European synchrotrons and a software development factory and, as such, defines a new paradigm for the development of beamline control platforms for the European MX user community.

Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis.

Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a π-π interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.

ID30B - a versatile beamline for macromolecular crystallography experiments at the ESRF.

ID30B is an undulator-based high-intensity, energy-tuneable (6.0-20 keV) and variable-focus (20-200 µm in diameter) macromolecular crystallography (MX) beamline at the ESRF. It was the last of the ESRF Structural Biology Group's beamlines to be constructed and commissioned as part of the ESRF's Phase I Upgrade Program and has been in user operation since June 2015. Both a modified microdiffractometer (MD2S) incorporating an in situ plate screening capability and a new flexible sample changer (the FlexHCD) were specifically developed for ID30B. Here, the authors provide the current beamline characteristics and detail how different types of MX experiments can be performed on ID30B (http://www.esrf.eu/id30b).

Multi-position data collection and dynamic beam sizing: recent improvements to the automatic data-collection algorithms on MASSIF-1.

Macromolecular crystallography is now a mature and widely used technique that is essential in the understanding of biology and medicine. Increases in computing power combined with robotics have not only enabled large numbers of samples to be screened and characterized but have also enabled better decisions to be taken on data collection itself. This led to the development of MASSIF-1 at the ESRF, the first beamline in the world to run fully automatically while making intelligent decisions taking user requirements into account. Since opening in late 2014, the beamline has processed over 42 000 samples. Improvements have been made to the speed of the sample-handling robotics and error management within the software routines. The workflows initially put into place, while highly innovative at the time, have been expanded to include increased complexity and additional intelligence using the information gathered during characterization; this includes adapting the beam diameter dynamically to match the diffraction volume within the crystal. Complex multi-position and multi-crystal data collections have now also been integrated into the selection of experiments available. This has led to increased data quality and throughput, allowing even the most challenging samples to be treated automatically.

Structural Evidence for a Role of the Multi-functional Human Glycoprotein Afamin in Wnt Transport.

Afamin, a human plasma glycoprotein and putative transporter of hydrophobic molecules, has been shown to act as extracellular chaperone for poorly soluble, acylated Wnt proteins, forming a stable, soluble complex with functioning Wnt proteins. The 2.1-Å crystal structure of glycosylated human afamin reveals an almost exclusively hydrophobic binding cleft capable of harboring large hydrophobic moieties. Lipid analysis confirms the presence of lipids, and density in the primary binding pocket of afamin was modeled as palmitoleic acid, presenting the native O-acylation on serine 209 in human Wnt3a. The modeled complex between the experimental afamin structure and a Wnt3a homology model based on the XWnt8-Fz8-CRD fragment complex crystal structure is compelling, with favorable interactions comparable with the crystal structure complex. Afamin readily accommodates the conserved palmitoylated serine 209 of Wnt3a, providing a structural basis how afamin solubilizes hydrophobic and poorly soluble Wnt proteins.

An algal photoenzyme converts fatty acids to hydrocarbons.

Although many organisms capture or respond to sunlight, few enzymes are known to be driven by light. Among these are DNA photolyases and the photosynthetic reaction centers. Here, we show that the microalga Chlorella variabilis NC64A harbors a photoenzyme that acts in lipid metabolism. This enzyme belongs to an algae-specific clade of the glucose-methanol-choline oxidoreductase family and catalyzes the decarboxylation of free fatty acids to n-alkanes or -alkenes in response to blue light. Crystal structure of the protein reveals a fatty acid-binding site in a hydrophobic tunnel leading to the light-capturing flavin adenine dinucleotide (FAD) cofactor. The decarboxylation is initiated through electron abstraction from the fatty acid by the photoexcited FAD with a quantum yield >80%. This photoenzyme, which we name fatty acid photodecarboxylase, may be useful in light-driven, bio-based production of hydrocarbons.

Structural insights into a family 39 glycoside hydrolase from the gut symbiont Bacteroides cellulosilyticus WH2.

Bacteria from the human gut are equipped with an arsenal of carbohydrate-active enzymes that degrade dietary and host-derived glycans. In this study, we present the 2.5Å resolution crystal structure of a member (GH39wh2) from the human gut bacteria Bacteroides cellulosilyticus WH2 representative of a new subgroup within family GH39. Together with 6 other GHs, GH39wh2 belongs to a polysaccharide utilization locus (PUL) that could be involved in detecting, binding and hydrolysing a specific carbohydrate species from the intestinal tract. GH39wh2 shares a similar architecture as other members of family GH39 dominated by a typical (ß/α)8-barrel fold harboring the catalytic residues and decorated by ß-sandwich accessory domains. The GH39wh2 structure unveils an atypical shallow groove rather than a deep pocket due to drastic rearrangements in surface loops surrounding the catalytic interface. These structural adaptations seem to favour recognition of large branched substrates and may explain the lack of activity of GH39wh2 toward small xylose-based and other typical substrates from GH39 members, emphasizing the molecular diversity within the GH39 family. A phylogenetic analysis of the entire GH39 family assigns GH39wh2 as a new subgroup, consistent with the extensive remodelling of the active site region that may confer new substrate specificity toward a complex glycan chain.

RoboDiff: combining a sample changer and goniometer for highly automated macromolecular crystallography experiments.

Automation of the mounting of cryocooled samples is now a feature of the majority of beamlines dedicated to macromolecular crystallography (MX). Robotic sample changers have been developed over many years, with the latest designs increasing capacity, reliability and speed. Here, the development of a new sample changer deployed at the ESRF beamline MASSIF-1 (ID30A-1), based on an industrial six-axis robot, is described. The device, named RoboDiff, includes a high-capacity dewar, acts as both a sample changer and a high-accuracy goniometer, and has been designed for completely unattended sample mounting and diffraction data collection. This aim has been achieved using a high level of diagnostics at all steps of the process from mounting and characterization to data collection. The RoboDiff has been in service on the fully automated endstation MASSIF-1 at the ESRF since September 2014 and, at the time of writing, has processed more than 20 000 samples completely automatically.

MASSIF-1: a beamline dedicated to the fully automatic characterization and data collection from crystals of biological macromolecules.

MASSIF-1 (ID30A-1) is an ESRF undulator beamline operating at a fixed wavelength of 0.969 Å (12.8 keV) that is dedicated to the completely automatic characterization of and data collection from crystals of biological macromolecules. The first of the ESRF Upgrade MASSIF beamlines to be commissioned, it has been open since September 2014, providing a unique automated data collection service to academic and industrial users. Here, the beamline characteristics and details of the new service are outlined.

Fully automatic characterization and data collection from crystals of biological macromolecules.

Considerable effort is dedicated to evaluating macromolecular crystals at synchrotron sources, even for well established and robust systems. Much of this work is repetitive, and the time spent could be better invested in the interpretation of the results. In order to decrease the need for manual intervention in the most repetitive steps of structural biology projects, initial screening and data collection, a fully automatic system has been developed to mount, locate, centre to the optimal diffraction volume, characterize and, if possible, collect data from multiple cryocooled crystals. Using the capabilities of pixel-array detectors, the system is as fast as a human operator, taking an average of 6 min per sample depending on the sample size and the level of characterization required. Using a fast X-ray-based routine, samples are located and centred systematically at the position of highest diffraction signal and important parameters for sample characterization, such as flux, beam size and crystal volume, are automatically taken into account, ensuring the calculation of optimal data-collection strategies. The system is now in operation at the new ESRF beamline MASSIF-1 and has been used by both industrial and academic users for many different sample types, including crystals of less than 20 µm in the smallest dimension. To date, over 8000 samples have been evaluated on MASSIF-1 without any human intervention.

Structural and biochemical characterization of the ß-N-acetylglucosaminidase from Thermotoga maritima: toward rationalization of mechanistic knowledge in the GH73 family.

Members of the GH73 glycosidase family cleave the ß-1,4-glycosidic bond between the N-acetylglucosaminyl (GlcNAc) and N-acetylmuramyl (MurNAc) moieties in bacterial peptidoglycan. A catalytic mechanism has been proposed for members FlgJ, Auto, AcmA and Atl(WM) and the structural analysis of FlgJ and Auto revealed a conserved α/ß fold reminiscent of the distantly related GH23 lysozyme. Comparison of the active site residues reveals variability in the nature of the catalytic general base suggesting two distinct catalytic mechanisms: an inverting mechanism involving two distant glutamate residues and a substrate-assisted mechanism involving anchimeric assistance by the C2-acetamido group of the GlcNAc moiety. Herein, we present the biochemical characterization and crystal structure of TM0633 from the hyperthermophilic bacterium Thermotoga maritima. TM0633 adopts the α/ß fold of the family and displays ß-N-acetylglucosaminidase activity on intact peptidoglycan sacculi. Site-directed mutagenesis identifies Glu34, Glu65 and Tyr118 as important residues for catalysis. A thorough bioinformatic analysis of the GH73 sequences identified five phylogenetic clusters. TM0633, FlgJ and Auto belong to a group of three clusters that conserve two carboxylate residues involved in a classical inverting acid-base mechanism. Members of the other two clusters lack a conserved catalytic general base supporting a substrate-assisted mechanism. Molecular modeling of representative members from each cluster suggests that variability in length of the ß-hairpin region above the active site confers ligand-binding specificity and modulates the catalytic mechanisms within the GH73 family.

Recent progress in robot-based systems for crystallography and their contribution to drug discovery.

INTRODUCTION: X-ray crystallography is the main tool for macromolecular structure solution at atomic resolution. It provides key information for the understanding of protein function, opening opportunities for the modulation of enzymatic mechanisms, and protein-ligand interactions. As a consequence, macromolecular crystallography plays an essential role in drug design, as well as in the a posteriori validation of drug mechanisms. AREAS COVERED: The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past 10 years. As a consequence, access to the facilities required for these investigations, such as synchrotron beamlines, became more difficult and significant efforts were dedicated to the automation of the experimental setup in laboratories. In this article, the authors describe how this was accomplished and how robot-based systems contribute to the enhancement of the macromolecular structure solution pipeline. EXPERT OPINION: The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community.

MxCuBE: a synchrotron beamline control environment customized for macromolecular crystallography experiments.

The design and features of a beamline control software system for macromolecular crystallography (MX) experiments developed at the European Synchrotron Radiation Facility (ESRF) are described. This system, MxCuBE, allows users to easily and simply interact with beamline hardware components and provides automated routines for common tasks in the operation of a synchrotron beamline dedicated to experiments in MX. Additional functionality is provided through intuitive interfaces that enable the assessment of the diffraction characteristics of samples, experiment planning, automatic data collection and the on-line collection and analysis of X-ray emission spectra. The software can be run in a tandem client-server mode that allows for remote control and relevant experimental parameters and results are automatically logged in a relational database, ISPyB. MxCuBE is modular, flexible and extensible and is currently deployed on eight macromolecular crystallography beamlines at the ESRF. Additionally, the software is installed at MAX-lab beamline I911-3 and at BESSY beamline BL14.1.

Circular permutation provides an evolutionary link between two families of calcium-dependent carbohydrate binding modules.

The microbial deconstruction of the plant cell wall is a critical biological process, which also provides important substrates for environmentally sustainable industries. Enzymes that hydrolyze the plant cell wall generally contain non-catalytic carbohydrate binding modules (CBMs) that contribute to plant cell wall degradation. Here we report the biochemical properties and crystal structure of a family of CBMs (CBM60) that are located in xylanases. Uniquely, the proteins display broad ligand specificity, targeting xylans, galactans, and cellulose. Some of the CBM60s display enhanced affinity for their ligands through avidity effects mediated by protein dimerization. The crystal structure of vCBM60, displays a ß-sandwich with the ligand binding site comprising a broad cleft formed by the loops connecting the two ß-sheets. Ligand recognition at site 1 is, exclusively, through hydrophobic interactions, whereas binding at site 2 is conferred by polar interactions between a protein-bound calcium and the O2 and O3 of the sugar. The observation, that ligand recognition at site 2 requires only a ß-linked sugar that contains equatorial hydroxyls at C2 and C3, explains the broad ligand specificity displayed by vCBM60. The ligand-binding apparatus of vCBM60 displays remarkable structural conservation with a family 36 CBM (CBM36); however, the residues that contribute to carbohydrate recognition are derived from different regions of the two proteins. Three-dimensional structure-based sequence alignments reveal that CBM36 and CBM60 are related by circular permutation. The biological and evolutionary significance of the mechanism of ligand recognition displayed by family 60 CBMs is discussed.

The ID23-2 structural biology microfocus beamline at the ESRF.

The first phase of the ESRF beamline ID23 to be constructed was ID23-1, a tunable MAD-capable beamline which opened to users in early 2004. The second phase of the beamline to be constructed is ID23-2, a monochromatic microfocus beamline dedicated to macromolecular crystallography experiments. Beamline ID23-2 makes use of well characterized optical elements: a single-bounce silicon (111) monochromator and two mirrors in Kirkpatrick-Baez geometry to focus the X-ray beam. A major design goal of the ID23-2 beamline is to provide a reliable, easy-to-use and routine microfocus beam. ID23-2 started operation in November 2005, as the first beamline dedicated to microfocus macromolecular crystallography. The beamline has taken the standard automated ESRF macromolecular crystallography environment (both hardware and software), allowing users of ID23-2 to be rapidly familiar with the microfocus environment. This paper describes the beamline design, the special considerations taken into account given the microfocus beam, and summarizes the results of the first years of the beamline operation.