Cryo2RT: a high-throughput method for room-temperature macromolecular crystallography from cryo-cooled crystals.

Advances in structural biology have relied heavily on synchrotron cryo-crystallography and cryogenic electron microscopy to elucidate biological processes and for drug discovery. However, disparities between cryogenic and room-temperature (RT) crystal structures pose challenges. Here, Cryo2RT, a high-throughput RT data-collection method from cryo-cooled crystals that leverages the cryo-crystallography workflow, is introduced.

Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp.

Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket.

Structure snapshots reveal the mechanism of a bacterial membrane lipoprotein -acyltransferase.

Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein -acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism.

Molecular basis of the key regulator WRINKLED1 in plant oil biosynthesis.

Vegetable oils are not only major components of human diet but also vital for industrial applications. WRINKLED1 (WRI1) is a pivotal transcription factor governing plant oil biosynthesis, but the underlying DNA-binding mechanism remains incompletely understood. Here, we resolved the structure of Arabidopsis WRI1 (AtWRI1) with its cognate double-stranded DNA (dsDNA), revealing two antiparallel β sheets in the tandem AP2 domains that intercalate into the adjacent major grooves of dsDNA to determine the sequence recognition specificity.

Probing ligand binding of endothiapepsin by `temperature-resolved' macromolecular crystallography.

Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed.

Biochemical, structural, and functional studies reveal that MAB_4324c from Mycobacterium abscessus is an active tandem repeat N-acetyltransferase.

Mycobacterium abscessus is a pathogenic non-tuberculous mycobacterium that possesses an intrinsic drug resistance profile. Several N-acetyltransferases mediate drug resistance and/or participate in M. abscessus virulence.

Structure-function relationship of a novel fucoside-binding fruiting body lectin from Coprinopsis cinerea exhibiting nematotoxic activity.

Lectins are non-immunoglobulin-type proteins that bind to specific carbohydrate epitopes and play important roles in intra- and inter-organismic interactions. Here, we describe a novel fucose-specific lectin, termed CML1, which we identified from fruiting body extracts of Coprinopsis cinerea. For further characterization, the coding sequence for CML1 was cloned and heterologously expressed in Escherichia coli.

Iterative Structure-Based Optimization of Short Peptides Targeting the Bacterial Sliding Clamp.

The bacterial DNA sliding clamp (SC), or replication processivity factor, is a promising target for the development of novel antibiotics. We report a structure-activity relationship study of a new series of peptides interacting within the SC (SC) binding pocket. Various modifications were explored including N-alkylation of the peptide bonds, extension of the N-terminal moiety, and introduction of hydrophobic and constrained residues at the C-terminus.

Tuning of the Berry curvature in 2D perovskite polaritons.

The engineering of the energy dispersion of polaritons in microcavities through nanofabrication or through the exploitation of intrinsic material and cavity anisotropies has demonstrated many intriguing effects related to topology and emergent gauge fields such as the anomalous quantum Hall and Rashba effects. Here we show how we can obtain different Berry curvature distributions of polariton bands in a strongly coupled organic-inorganic two-dimensional perovskite single-crystal microcavity. The spatial anisotropy of the perovskite crystal combined with photonic spin-orbit coupling produce two Hamilton diabolical points in the dispersion.

Managing Growth and Dimensionality of Quasi 2D Perovskite Single-Crystalline Flakes for Tunable Excitons Orientation.

Hybrid perovskites are among the most promising materials for optoelectronic applications. Their 2D crystalline form is even more interesting since the alternating inorganic and organic layers naturally forge a multiple quantum-well structure, leading to the formation of stable excitonic resonances. Nevertheless, a controlled modulation of the quantum well width, which is defined by the number of inorganic layers (n) between two organic ones, is not trivial and represents the main synthetic challenge in the field.

Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology.

Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible and therefore unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH), can be seamlessly fused to terminal helices of proteins, forming an extended, partially free-standing rigid helix.

The crystal structure of MreC provides insights into polymer formation.

MreC is a scaffold protein required for cell shape determination through interactions with proteins related to cell wall synthesis. Here, we determined the crystal structure of the major periplasmic part of MreC from Escherichia coli at 2.1 Å resolution.

Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis.

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit.

Crystal structure of SARS-CoV-2 Orf9b in complex with human TOM70 suggests unusual virus-host interactions.

Although the accessory proteins are considered non-essential for coronavirus replication, accumulating evidences demonstrate they are critical to virus-host interaction and pathogenesis. Orf9b is a unique accessory protein of SARS-CoV-2 and SARS-CoV. It is implicated in immune evasion by targeting mitochondria, where it associates with the versatile adapter TOM70.

Structural Insights into Plasticity and Discovery of Remdesivir Metabolite GS-441524 Binding in SARS-CoV-2 Macrodomain.

The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in the host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus targeting this protein domain may offer a therapeutic strategy to combat current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally occurring nucleotides, small molecules, and nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir.

Drosophila TNFRs Grindelwald and Wengen bind Eiger with different affinities and promote distinct cellular functions.

The Drosophila tumour necrosis factor (TNF) ligand-receptor system consists of a unique ligand, Eiger (Egr), and two receptors, Grindelwald (Grnd) and Wengen (Wgn), and therefore provides a simple system for exploring the interplay between ligand and receptors, and the requirement for Grnd and Wgn in TNF/Egr-mediated processes. Here, we report the crystallographic structure of the extracellular domain (ECD) of Grnd in complex with Egr, a high-affinity hetero-hexameric assembly reminiscent of human TNF:TNFR complexes. We show that ectopic expression of Egr results in internalisation of Egr:Grnd complexes in vesicles, a step preceding and strictly required for Egr-induced apoptosis.

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme.

Engineering the Optical Emission and Robustness of Metal-Halide Layered Perovskites through Ligand Accommodation.

The unique combination of organic and inorganic layers in 2D layered perovskites offers promise for the design of a variety of materials for mechatronics, flexoelectrics, energy conversion, and lighting. However, the potential tailoring of their properties through the organic building blocks is not yet well understood. Here, different classes of organoammonium molecules are exploited to engineer the optical emission and robustness of a new set of Ruddlesden-Popper metal-halide layered perovskites.

WDR90 is a centriolar microtubule wall protein important for centriole architecture integrity.

Centrioles are characterized by a nine-fold arrangement of microtubule triplets held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood.

Synthesis, crystal structure, polymorphism and microscopic luminescence properties of anthracene derivative compounds.

Anthracene derivative compounds are currently investigated because of their unique physical properties (e.g. bright luminescence and emission tunability), which make them ideal candidates for advanced optoelectronic devices.

3D-printed holders for fixed-target serial X-ray crystallography.

The serial X-ray crystallography method was developed to ease the handling of small fragile crystals of membrane proteins and for rapid data collection on hundreds of microcrystals directly in the growth medium without the need for crystal harvesting. To facilitate mounting of these samples on a goniometer at cryogenic or at room temperatures, two new 3D-printed holders have been developed. They provide for cubic and sponge phase sample stability in the X-ray beam and are compatible with sample-changing robots.

The TELL automatic sample changer for macromolecular crystallography.

In this paper, the design and functionalities of the high-throughput TELL sample exchange system for macromolecular crystallography is presented. TELL was developed at the Paul Scherrer Institute with a focus on speed, storage capacity and reliability to serve the three macromolecular crystallography beamlines of the Swiss Light Source, as well as the SwissMX instrument at SwissFEL.

The human telomeric nucleosome displays distinct structural and dynamic properties.

Telomeres protect the ends of our chromosomes and are key to maintaining genomic integrity during cell division and differentiation. However, our knowledge of telomeric chromatin and nucleosome structure at the molecular level is limited. Here, we aimed to define the structure, dynamics as well as properties in solution of the human telomeric nucleosome.

In Meso In Situ Serial X-Ray Crystallography (IMISX): A Protocol for Membrane Protein Structure Determination at the Swiss Light Source.

The lipid cubic phases (LCP) have enabled the determination of many important high-resolution structures of membrane proteins such as G-protein-coupled receptors, photosensitive proteins, enzymes, channels, and transporters. However, harvesting the crystals from the glass or plastic plates in which crystals grow is challenging. The in meso in situ serial X-ray crystallography (IMISX) method uses thin plastic windowed plates that minimize LCP crystal manipulation.