RNA oligomers at atomic resolution containing 1-methylpseudouridine, an essential building block of mRNA vaccines.

All widely used mRNA vaccines against COVID-19 contain in their sequence 1-methylpseudouridine (m1Ψ) instead of uridine. In this publication, we report two high resolution crystal structures (at up to 1.01 and 1.32 Å, respectively) of one such double-stranded 12-mer RNA sequence crystallized in two crystal forms. The structures are compared with similar structures which do not contain this modification. Additionally, the X-ray structure of 1-methyl-pseudouridine itself was determined.

Micro-structured polymer fixed targets for serial crystallography at synchrotrons and XFELs.

Fixed targets are a popular form of sample-delivery system used in serial crystallography at synchrotron and X-ray free-electron laser sources. They offer a wide range of sample-preparation options and are generally easy to use. The supports are typically made from silicon, quartz or polymer. Of these, currently, only silicon offers the ability to perform an aperture-aligned data collection where crystals are loaded into cavities in precise locations and sequentially rastered through, in step with the X-ray pulses. The polymer-based fixed targets have lacked the precision fabrication to enable this data-collection strategy and have been limited to directed-raster scans with crystals randomly distributed across the polymer surface. Here, the fabrication and first results from a new polymer-based fixed target, the micro-structured polymer fixed targets (MISP chips), are presented. MISP chips, like those made from silicon, have a precise array of cavities and fiducial markers. They consist of a structured polymer membrane and a stabilization frame. Crystals can be loaded into the cavities and the excess crystallization solution removed through apertures at their base. The fiducial markers allow for a rapid calculation of the aperture locations. The chips have a low X-ray background and, since they are optically transparent, also allow for an a priori analysis of crystal locations. This location mapping could, ultimately, optimize hit rates towards 100%. A black version of the MISP chip was produced to reduce light contamination for optical-pump/X-ray probe experiments. A study of the loading properties of the chips reveals that these types of fixed targets are best optimized for crystals of the order of 25 µm, but quality data can be collected from crystals as small as 5 µm. With the development of these chips, it has been proved that polymer-based fixed targets can be made with the precision required for aperture-alignment-based data-collection strategies. Further work can now be directed towards more cost-effective mass fabrication to make their use more sustainable for serial crystallography facilities and users.

High-Throughput Crystallography Reveals Boron-Containing Inhibitors of a Penicillin-Binding Protein with Di- and Tricovalent Binding Modes.

The effectiveness of ß-lactam antibiotics is increasingly compromised by ß-lactamases. Boron-containing inhibitors are potent serine-ß-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) ß-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by ß-lactamase-driven resistance.

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme.

This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97-Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.

How best to use photons.

Strategies for collecting X-ray diffraction data have evolved alongside beamline hardware and detector developments. The traditional approaches for diffraction data collection have emphasised collecting data from noisy integrating detectors (i.e. film, image plates and CCD detectors). With fast pixel array detectors on stable beamlines, the limiting factor becomes the sample lifetime, and the question becomes one of how to expend the photons that your sample can diffract, i.e. as a smaller number of stronger measurements or a larger number of weaker data. This parameter space is explored via experiment and synthetic data treatment and advice is derived on how best to use the equipment on a modern beamline. Suggestions are also made on how to acquire data in a conservative manner if very little is known about the sample lifetime.

Where is crystallography going?

Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide.

Application of in situ diffraction in high-throughput structure determination platforms.

Macromolecular crystallography (MX) is the most powerful technique available to structural biologists to visualize in atomic detail the macromolecular machinery of the cell. Since the emergence of structural genomics initiatives, significant advances have been made in all key steps of the structure determination process. In particular, third-generation synchrotron sources and the application of highly automated approaches to data acquisition and analysis at these facilities have been the major factors in the rate of increase of macromolecular structures determined annually. A plethora of tools are now available to users of synchrotron beamlines to enable rapid and efficient evaluation of samples, collection of the best data, and in favorable cases structure solution in near real time. Here, we provide a short overview of the emerging use of collecting X-ray diffraction data directly from the crystallization experiment. These in situ experiments are now routinely available to users at a number of synchrotron MX beamlines. A practical guide to the use of the method on the MX suite of beamlines at Diamond Light Source is given.

Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging.

The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

Automated data collection for macromolecular crystallography.

An overview, together with some practical advice, is presented of the current status of the automation of macromolecular crystallography (MX) data collection, with a focus on MX beamlines at Diamond Light Source, UK.

Structural basis of gate-DNA breakage and resealing by type II topoisomerases.

Type II DNA topoisomerases are ubiquitous enzymes with essential functions in DNA replication, recombination and transcription. They change DNA topology by forming a transient covalent cleavage complex with a gate-DNA duplex that allows transport of a second duplex though the gate. Despite its biological importance and targeting by anticancer and antibacterial drugs, cleavage complex formation and reversal is not understood for any type II enzyme. To address the mechanism, we have used X-ray crystallography to study sequential states in the formation and reversal of a DNA cleavage complex by topoisomerase IV from Streptococcus pneumoniae, the bacterial type II enzyme involved in chromosome segregation. A high resolution structure of the complex captured by a novel antibacterial dione reveals two drug molecules intercalated at a cleaved B-form DNA gate and anchored by drug-specific protein contacts. Dione release generated drug-free cleaved and resealed DNA complexes in which the DNA gate instead adopts an unusual A/B-form helical conformation with a Mg(2+) ion repositioned to coordinate each scissile phosphodiester group and promote reversible cleavage by active-site tyrosines. These structures, the first for putative reaction intermediates of a type II topoisomerase, suggest how a type II enzyme reseals DNA during its normal reaction cycle and illuminate aspects of drug arrest important for the development of new topoisomerase-targeting therapeutics.