Wednesday 6th- Thursday 7th November 2024​
Talks
Study of phosphate uptake mechanism by a prominent human gut symbiont
Dr Yiling Zhu, Newcastle University
Phosphorus is an essential mineral, and phosphate is a key component of cellular structures such as membranes, DNA, RNA, and ATP, the cell’s energy currency. In the human gut microbiota, phosphorus acts as a major nutrient, influencing microbial activity, composition, and distribution. Gut bacteria that store phosphate can potentially benefit the host by reducing the phosphate burden on the gut epithelium. In this study, we investigate the phosphate uptake channels of Bacteroides thetaiotaomicron, a prominent human gut symbiont. Through proteomic analysis, we identified three potential phosphate uptake complexes that were overexpressed under phosphate-limited conditions. Each of these systems includes an outer membrane TonB-dependent transporter and a cell surface-exposed lipoprotein. We determined the crystal structures of the lipoproteins and obtained a single-particle cryo-EM structure of one of the complexes. To further understand substrate specificity, single, double, and triple knockouts of the three systems were generated. Our work demonstrates the structural diversity and specificity of phosphate uptake mechanisms in B. thetaiotaomicron, highlighting its potential role in modulating gut phosphorus levels and contributing to host-microbiota interactions. These findings provide novel insights into the nutrient acquisition strategies of gut bacteria and open avenues for future research into the manipulation of gut symbionts for host health benefits.
Uncovering the role of stereoisomerism in the enzymatic
mechanism of a novel alginate lyase.
William Houppy, Northumbria University
Alginate lyases (AL) are enzymes capable of depolymerizing alginate, a polysaccharide extracted from seaweed. These enzymes have gained increasing importance in various industries, including food production, laundry detergence, and biomass valorization. In this study, two efficient ALs from the CAZy PL7 family, identified in Flavobacterium species, were analyzed through a mechanistic study to explain the observed variations in activity.
Alginate consists of two monomers, β-á´…-mannuronate (M) and its C5 epimer α-ÊŸ-guluronate (G), connected through β-1,4 glycosidic bonds. Comparative analysis of one of the ALs (Aly30) with other previously described ALs revealed a conserved structure, using an AlphaFold prediction, and a similar active site centred around a histidine (HIS) and a tyrosine (TYR) residues.
Typically, ALs follow a β-elimination mechanism, where HIS acts as a base and TYR as an acid, attacking the α-hydrogen at the C5 position. We have studied Aly30 with the help of docking in regards to poly-G and poly-M, the latter its preferential substrate. However, for poly-M, docking did not support this initial hypothesis, indicating an alternative role for the tyrosine.
As this was further supported by molecular dynamic studies, we propose an alternative mechanism where TYR in the active site plays a distinct role, especially considering that TYR can act in its deprotonated state. Since only difference between M and G is the configuration of the C5 position, the stereoisomerism of the substrate appears to dictate the enzymatic mechanism which may explain the observed variations in AL activity.
We are currently validating our computational findings with additional wet-lab experiments and conducting quantum chemistry studies to draw the energy profiles of the proposed mechanisms.
Cryo-EM structures of pseudouridine synthase 3 and tRNA complex reveal the regulation mechanism
Yasmin Stone, Durham University
RNA modifications expand the structural and functional capacities of RNAs. Pseudouridine, an isomer of uridine, is found in various RNA classes to attenuate RNA structure, thermostability, and RNA-protein interactions, affecting RNA metabolism and fine-tuning protein translation machinery. Human pseudouridine synthases 3 (PUS3) is responsible for pseudouridine conversion on tRNAs. Recently, several case studies reported the link of PUS3 variants to neurodegenerative diseases. Although characterisations of the bacterial counterpart of PUS3 are already well-documented, the intensive investigation of human PUS3 has just begun. First, we obtained the recombinantly expressed human PUS3 using an insect cell expression system. We characterise its biochemical activity towards various tRNAs, including precursor and mature versions, and also present the first cryo-EM structures of human PUS3 with tRNA substrates at multiple resolutions (2.8-3.3 Å). Second, although the overall PUS3/tRNA complex structure resembles that of bacterial homolog, we identify that PUS3 utilises the unique intertwined long helices from the C-termini of each monomer for the dimer formation. In addition, the elbow of tRNA seems to serve as the contact determination point. Third, combining local high-resolution structure and 3D variable analysis shows the local movement of ASL, hinting at a dynamic interaction of ASL and the catalytic residue and other regulatory residues during the reaction. Last, biochemical and cellular characterisations of clinical-derived PUS variants explain how the mutations impede enzyme activity, leading to cellular dysregulation. Our study expands our knowledge of the long-neglected protein and its importance in cells.
How to collect rotation data from <20 μm crystals at the VMXm beamline
Dr Adam Crawshaw, Diamond Light Source
The Versatile Macromolecular Crystallography Microfocus (VMXm) beamline is a nano-/micro-focus beamline at Diamond Light Source which has been optimised for the collection of rotation X-ray diffraction data from crystals measuring 1-20 μm in size. The design of the beamline is centered around the minimization of background scatter, to ensure measurement of the weak diffraction generated by microcrystals. The beamline optics deliver a beam of 0.4 - 9 μm vertically and 1.3 - 13 μm horizontally at the sample position across an energy range of 10-22 keV, delivering ~10^10 ph/s to the sample at 21.3 keV. The beamline is equipped with an Eiger2 X 9M CdTe which enables data collection at higher X-ray energies (>18 keV) which can extend the life of a crystal in the beam, allowing more information to be recorded, as well increasing the diffraction signal.​
Microcrystals are prepared on electron microscopy grids using techniques borrowed from cryoEM, including grid blotting and plunge cooling in liquid ethane. Samples are prepared in advance using the VMXm support laboratory with the assistance of beamline staff. Once grids are introduced to the beamline, crystals can be visualized and aligned to the X-ray beam using either an on-axis optical microscope or the integrated SEM. Signal-to-noise of the diffracted X-rays is greatly improved due to the mounting technique of the crystals which are held under vacuum for diffraction measurements, reducing background scatter to a minimum.
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The talk will describe how to best collect rotation data from microcrystals using VMXm and how the beamline can be accessed. Several case studies will be described, including a novel structure solved using Se derivatized samples, a GPCR membrane protein, needle/sea urchin crystals as well as the application of ligand binding with microcrystals.
Structural characterisation of aminoadipate semialdehyde synthase, a novel therapeutic target for lysine metabolic disorders
Dr Richard Heath, Newcastle University
There are two early onset metabolic disorders in humans caused by malfunctions in lysine catabolism: Pyridoxine dependant epilepsy (PDE), caused by lack of enzyme ALDH7A1, resulting in toxic levels of the metabolite, aminoadipate semialdehyde (α-AASA); and Glutaric aciduria type 1 (GA1), caused by inherited deficiency of glutaryl-CoA dehydrogenase. The symptoms of either disorder can be alleviated by switching off lysine degradation. The potential therapeutic target for such ‘substrate reductive therapy’ is the first enzyme in the lysine metabolic pathway, aminoadipate semialdehyde synthase (AASS).
The bifunctional enzyme AASS consists of the lysine ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH) domains. SDH converts saccharopine to α-AASA and as such is the domain responsible for the toxic build-up of substrate in PDE. High throughput screening (HTS) identified hits for SDH, which were validated in two enzymatic assays as well as binding assays (SPR) and cellular target engagement assays (CETSA). Crystal structures of SDH with several lead compounds demonstrated different binding modes within the substrate binding pocket, enabling merged compounds to be made with better potency.
Direct targeting of the SDH may not therapeutically beneficial, based on emerging evidence that build-up of saccharopine may be detrimental. An alternative approach is to target the LKR domain, which converts lysine to saccharopine. We have a 3.5 Å crystal structure of LKR and a 4 Å Cryo-EM reconstruction of full length AASS. The 2D classes reveal the architecture of full length AASS: a tetrameric LKR core with SDH dimers either side. We are currently embarking on screening approaches such as ASMS and HTS to identify hits for LKR which can be further developed by structure-based drug design. In summary, our integrated structural approach on both domains enables future efforts to identify therapeutics for PDE and GA1.
NucleoFind: a deep-learning network for interpreting nucleic acid electron density
Jordan Dialpuri, University of York
Nucleic acid electron density interpretation after phasing by molecular replacement or other methods remains a difficult problem for computer programs to deal with. Programs tend to rely on time-consuming and computationally exhaustive searches to recognise characteristic features. We present NucleoFind, a deep-learning-based approach to interpreting and segmenting electron density. Using an electron density map from X-ray crystallography obtained after molecular replacement, the positions of the phosphate group, sugar ring and nitrogenous base group can be predicted with high accuracy. On average, 78% of phosphate atoms, 85% of sugar atoms and 83% of base atoms are positioned in predicted density after giving NucleoFind maps produced following successful molecular replacement. NucleoFind can use the wealth of context these predicted maps provide to build more accurate and complete nucleic acid models automatically.
New Capabilities at Diamond Beamline VMXi
Dr Amy Thompson, Diamond Light Source
Room temperature crystallography is undergoing a resurgence as automatic data processing pipelines now have the ability to keep up with the multi-crystal strategies required to avoid radiation damaged structures. To enable room temperature data collection, the Diamond Light Source beamline VMXi was built to measure in-situ samples at a high-throughput rate. By implementing automatic crystal finding algorithms (CHIMP), as well as processing pipelines for merging multi-crystal datasets (xia2.multiplex), VMXi achieves a high level of automation capable of providing high-quality data, even for traditionally challenging samples. Recent developments have focused on new capabilities in dataset clustering, fragment screening and serial crystallography. Improvements to xia2.multiplex will be presented, demonstrating the ability to quickly identify subtle differences between large numbers of datasets. These improvements in the clustering algorithm could be used to identify differences such as conformational changes or bound fragments. Ligands have also been demonstrated to bind differently under room and cryogenic conditions. To meet a growing need to compare the results of drug-discovery campaigns at both temperatures, fragment screening pipelines are also being developed at VMXi. Finally, the ability to quickly screen batch crystallisation samples for serial crystallography will be demonstrated, as a method to improve crystal homogeneity and quality with minimal sample loss.
Determining the structure of the CDK2-cyclin A-CDC25A complex by Cryo-EM
Dr Rhianna Rowland, Newcastle University
The cell division cycle 25 phosphatases CDC25A, B and C regulate the eukaryotic cell cycle by removing the inhibitory phosphorylation on cyclin-dependent protein kinases (CDKs) to activate CDK activity at cell-cycle checkpoints. Both CDC25A and B are oncogenes, frequently overexpressed in a variety of cancers with poor prognosis. Consequently, the CDC25 isoforms are attractive anticancer drug targets. However, until this work, no structure of a CDC25 phosphatase in complex with a CDK-cyclin module existed, hindering the development of CDC25-active compounds. We determined the structure of the 86 kDa CDC25A-CDK2-cyclin A complex to 2.7 Å resolution by cryogenic electron microscopy (cryo-EM). This structure reveals the overall complex architecture, detailing key protein-protein interactions that underpin CDC25A binding. We further identify a previously unobserved CDC25A C-terminal helix, that binds across the CDK2-cyclin A interface and is critical for complex formation. Comparisons with existing structures of CDK2 bound to kinase-associated phosphatase and cyclin-dependent protein kinase subunit 1 indicate that the CDK GDSEID motif (single letter amino acid code) is an important mutual binding site for these proteins that also imparts selectivity. Complementary sequence conservation and AlphaFold analysis, suggest CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whereas CDK4/6-cyclin D complexes appear unlikely substrates. We envisage this cryo-EM structure will improve our understanding of CDC25-dependent regulation of CDK activity, whilst facilitating the identification of allosteric sites amenable to the development of inhibitors to disrupt complex formation and CDC25 catalysis.
Structure and functional characterisation enzyme involved in Chagas disease
Sagar Batra, University of Nottingham
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a significant health concern in Latin America. A potential breakthrough in Chagas disease vaccine development lies in targeting Trypanosoma cruzi 80kDa prolyl oligopeptidase (TcPOP), a validated antigen. In this study, TcPOP was expressed in Escherichia coli, purified, and utilized as an immunogen in mice to investigate the resulting immune response.
Recombinant TcPOP was successfully purified to homogeneity and analysed using SEC-MALS and SEC-SAXS. Secondly, the purified TcPOP was used to immunise mice in conjugation with an adjuvant, eliciting a robust immune response. Monoclonal and polyclonal antibodies were purified from immunized mice using hybridoma technology and assessed for their reactivity against TcPOP and exhibited a strong and specific response confirmed using Enzyme-Linked Immunosorbent Assay (ELISA), BLI and Mass photometry. Despite sub-80 kDa size of TcPOP, the antigen was employed on cryoEM grids to be screened under the cryoTEM.
I collected data on Krios 300 KeV and after processing the dataset I was able to resolve the structure of apo-Tc80 at 2.5-3Å resolution. Interesting, I found that the structure exhibits multiple conformation specifically closed, and open states, therefore, I was able to deduce both the conformation at 3Å resolution and determine the secondary structure elements. This outcome demonstrated the structural variability and dynamics of not just TcPOP but the family of prolyl oligopeptidases, and the immunogenicity of TcPOP and its potential as a vaccine candidate. Mice immunization with TcPOP yielded promising results.
Future research endeavours will focus on utilizing the information from the TcPOP-mAb complex, to later pin down the epitope that could be crucial for antigen recognition and parasite invasion. These structural insights will provide valuable information for rational vaccine design and therapeutic development against Chagas disease.
Exploiting Anomalous Signal - Long Wavelength Macromolecular Crystallography at Beamline I23
Dr Christian Orr, Diamond Light Source
Beamline I23, a unique long-wavelength, in-vacuum macromolecular crystallography beamline, plays a crucial role in resolving the crystallographic phase problem and identifying atoms of biological relevance such as sulfur, phosphorus, chlorine, potassium, and calcium. By generating anomalous difference Fourier maps at wavelengths above and below the absorption edges of elements, we can precisely identify specific ions in their exact locations.
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While Alphafold has transformed structural biology, SAD phasing remains essential for challenging cases, such as proteins from thermophilic, psychrophilic, and halophilic organisms. I23 determines phases directly from native atoms within the protein, eliminating the need for SelMet substitution or heavy atom soaking, both of which are known to negatively impact crystal quality.
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A major limitation for long-wavelength crystallography comes from increased X-ray absorption by the sample. We have made progress to overcome these with recent developments in absorption correction methods, enabling the detection of lighter elements such as magnesium and sodium. A femtosecond laser enables the precise ablation of material surrounding the crystal, such as the loop and solvent, with minimal heat generation and damage. This technique allows for the reshaping of crystals into spheres, reducing the reliance on mathematical absorption corrections. Alternatively, tomographic reconstructions of the sample, solvent, and loop are used to ray trace reflections through material and apply absorption corrections analytically in a recently developed program, AnACor.
Beamline I23 is accessible through Rapid Access or your BAG allocation. Experiments are straightforward: sample mounts are shipped directly to your lab for crystal harvesting, and comprehensive training is provided for all skill levels, both on-site at Diamond and remotely.
Quantitative structural heterogeneity analysis of SARS-CoV-2 Spike protein in the context of the Omicron BA.2 variant and the D614G mutant
Dr James Krieger, CNB-CSIC
SARS-CoV-2 is the causative agent of COVID-19. Infection of cells by SARS-CoV-2 is driven by engagement of the trimeric viral spike protein with the cellular receptor ACE2 via its receptor-binding domain (RBD) and with other co-receptors via the N-terminal domain (NTD), both of which are also targets for neutralizing antibodies. The spike protein is a dynamic trimeric protein assembly, but cryo-EM analyses to date have principally resulted in classification of the structure of the complex into discrete states, and not focused on recovering the dynamic aspects directly from the cryo-EM images.
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In this work we present a robust, quantitative approach to analyze the conformational spread in the spike protein. We apply this analysis to the BA.2 Omicron variant, contrasting its properties with that of a mutant (D614G) that emerged early in the pandemic and did not yet have the mutations that appeared during the course of the pandemic. Our results show that the BA.2 spike protein is significantly more flexible than the D614G mutant and reveal unique states with greater extents of RBD compaction and closure and the NTD more tightly “locked” onto them as compared to the D614G mutant. We suggest that this difference in conformational spectrum may have been one of the factors that contributed to the more rapid spread of the BA.2 variant and other viral strains that followed the D614G mutant.
Structural and functional analysis of split DNA ligases from T5-like bacteriophages
Lu Zhang, University of Edinburgh
T5-like bacteriophages, which infect Gram-negative bacteria, have persistent single-stranded nicks at the 5′-GCGC sequence in their genomes. They encode an unusual NAD+-dependent heterodimeric DNA ligase composed of A and B subunits. This project aims to discover, using structural and biochemical methods, how these subunits function together and separately, and whether the split architecture relates to the genomic nicks.
Solution structural analysis by small-angle X-ray scattering (SAXS) shows that the split DNA ligase has an extended and flexible structure in the absence of DNA, similar to E. coli LigA (EcoLigA). X-ray crystallographic analysis of the PR1 (T5-like) DNA ligase bound to nicked dsDNA reveals that the A and B subunits encircle the DNA similarly to EcoLigA and provides insight into the role of the C-terminal BRCT domain. Interactions between the ligase and DNA are non-sequence-specific, consistent with biochemical assays – no preference for the ligation of random sequences over 5’-GCGC.
Expression of the A and B subunits is controlled by three promoters, potentially leading to unequal amounts of each subunit. Excess B-subunit decreases ligation efficiency. Electrophoresis mobility shift assays show that the B-subunit binds independently to dsDNA, with a preference for nicked and 5’-GCGC DNA. Structural models of the T5B-DNA complex, validated by SEC-SAXS, reveal interactions between all three domains of the B-subunit and DNA. Microscale Thermophoresis using B-subunit mutants identifies key residues involved in recognising the nicked DNA and 5’-GCGC sequence, suggesting the BRCT domain recognises phage genome nicks.
A molecular mechanism is proposed for the split DNA ligase in ligation, whereby the B-subunit first contacts DNA via the BRCT domain, allowing the catalytic A-subunit to bind and ligate the nick. Excess B-subunit preferentially binds and protects 5’-GCGC nicks, contributing to their persistence in the phage genome.
Mass Spectrometry for Structural Biology.
Dr Sally Shirran, University of St Andrews
An exploration of the role of mass spectrometry (MS) in supporting and advancing structural biology. Established techniques such as protein identification, confirmation of post-translational modifications, intact protein measurement, and limited proteolysis have long been integral to the field. However, recent advancements in sample preparation have broadened the applicability of these methods to a wider array of protein samples, including membrane proteins.
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Moreover, innovations in instrumentation have enhanced the versatility of mass spectrometry, allowing it to become an increasingly vital component of the structural biology toolkit. MS now provides complementary data alongside traditional techniques such as X-ray crystallography, NMR, and cryo-electron microscopy. Techniques including native mass spectrometry, affinity purification mass spectrometry, and cross-linking mass spectrometry yield distinct structural insights, offering information on composition and stoichiometry, as well as residue-level proximity and solvent accessibility.
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This presentation will discuss these techniques in detail, supported by case studies that illustrate their impact on recent structural investigations. Notable examples include a multiplexed mass spectrometry approach for the in depth characterization of the exosome complex from Saccharomyces cerevisiae expressed at physiologically relevant levels; structural analysis of leader sequence peptides derived from cyanobactins; and elucidation of how the bacterial cyclic oligonucleotide-based antiphage signaling system (CBASS) employs its intrinsic protein modification system to regulate nucleotide cyclase activity.
Using Cryo-EM and AlphaFold to Survey the Dynamic Landscape and Interactions of a Vitamin B12-utilising Protein Machine
Douglas Ferreira, Newcastle University
Isolated remethylation disorders are inborn errors of cobalamin (Cbl) metabolism that disrupt homocysteine homeostasis. They can result from pathogenic variants in methionine synthase (MTR, CblG defect), the Cbl-dependent enzyme that catalyses the remethylation of homocysteine into methionine; the MMADHC protein (CblD-HC defect) that delivers the Cbl cofactor to MTR; and the MTRR protein (CblE defect) that modifies Cbl to the usable form for MTR. How these proteins function with each other, and how these protein-protein interactions are compromised in disease-causing mutations, are unknown.
We set out to determine the interaction of MTR with MTRR and MMADHC using computational structural biology. We generated recombinant active MTR, determined its 3D structure using cryo-electron microscopy (cryo-EM) and modelled conformation dynamics of MTR in complex with MTRR and MMADHC using AlphaFold (AF2)-Multimer.
Our cryo-EM study, the first for human MTR, reveals a highly dynamic five-domain architecture. MTR exhibits a predominant inter-domain conformation, that is predicted by AF2 modelling to interact with MTRR and MMADHC at the same time. This conformation therefore enables MTR to receive the Cbl cofactor from CblD, and for Cbl to be modified by MTRR, as a three-way comple. Importantly, a number of disease-causing variants map to the MTR-MTRR-MMADHC interfaces, implicating a disruption in the complex as part of the patho-mechanism.
Our findings illustrate the complex choreography required for the Cbl loading and activation steps that drive MTR catalysis. This work exemplifies how experimental (cryo-EM) and computational (AF2) structural biology emerges as an integrated toolkit in understanding structural features of variants that lead to disruption of protein-protein interactions, setting the stage for elucidating other complexes in Cbl metabolism.
Novel predatory-specific lipid-binding transport in Bdellovibrio bacteriovorus
Stella Sultan, University of Birmingham
Bdellovibrio bacteriovorus (Bdellovibrio) is a Gram-negative predatory bacteria that preys on other Gram-negative bacteria. Due to its broad range of prey, there is potential for Bdellovibrio to be used as a living antibiotic. In its life cycle, Bdellovibrio swims freely in search of prey, invades through the outer membrane, and consumes the host cell from within, generating new progeny in the process.
Among the genes upregulated upon entry to prey, several contain a lipobox at the beginning of their sequence, showing the tethering of these proteins to the cell envelope. Based on AlphaFold predictions, two homologous proteins of different sizes have a novel fold that potentially binds to lipids. These hint at a possible function as an external lipid transport bridge, connecting Bdellovibrio with its prey in the predation process.
One of the proteins, Bdellovibrio envelope lipid transporter 1 (Belt1), was successfully extracted with detergent, in a similar manner to membrane protein treatment. When the initial hydrophobic residues were removed, the protein expressed solubly and was successfully crystallised. TLC confirmed the lipid-binding property of the proteins, demonstrating specificity for cardiolipin and phosphatidylglycerol. While initial crystals only diffracted to 7 Å, diffraction data from tiny crystals (5-7 µm) obtained with the VMXm beamline at Diamond Light Source resulted in a 2.29 Å structure of Belt1.
We discovered a new family of lipid binding protein represented by Belt1 and Belt2, which are entirely constructed of alpha-helices. X-ray crystallography data of Belt1 show lipids bound in the hydrophobic cavity of the protein, while the CryoEM data of Belt2 is currently being processed. This is an exciting discovery as all previously established lipid binders are made of beta-sheets. The characterisation of Belt1 and Belt2 and their unique architecture would potentially give clues to a predator-specific lipid transport system.
Understanding the use of novel macrocyclic peptides as substrates and inhibitors of SARS-CoV-2 Mpro
Taylah Andrews-Clark, University of Oxford/Diamond Light Source
The COVID-19 pandemic caused by the virus SARS-CoV-2, has had an unprecedented impact on public health and the global economy since its inception in late 2019. A key protein of SARS-CoV-2 is a cysteine protease, referred to as the main protease (Mpro). Mpro is essential for viral replication; In the early stages of the viral lifecycle Mpro cleaves the two polyproteins pp1a and pp1ab to release 16 non-structural proteins (nsp’s) that facilitate the assembly of new virions and proliferation of SARS-CoV-2 during infection. For this reason, we aim to study Mpro, and other related viral proteases, using time-resolved crystallography to improve our understanding of the chemical mechanisms involved in proteolytic cleavage to provide deeper insights that may aid the development of novel strategies for future antiviral discovery. Here, we describe the design of a novel cyclic peptide substrate, TAC-NSP-11, and the optimisation of microcrystal preparations that will aid in this goal.
New possibilities at I24: from cryo MX to time-resolved serial crystallography
Dr Sofia Jaho, Diamond Light Source
I24 is a microfocus beamline for macromolecular crystallography (MX) offering a fully tunable energy range, high flux density and adaptable beam size. Even though originally designed to serve users for conventional pin-based cryo MX, I24 has grown over the years to a versatile beamline exploring advanced developments in the field and other modes for data collection under room temperature conditions. Currently, the beamline features a modular sample environment designed in a plug-and-play format: pin-based cryo MX experiments and fixed-target serial data collection can be performed within the same shift requiring only a few minutes for setting up.
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Time-resolved X-ray crystallography using the serial approach has received increased attention over the past years, not only at XFELs but also at synchrotron sources (SSX). I24 has been leading developments in instrumentation, data collection strategies and data processing lowering the entry barrier to a wider user community. Serial experiments can be performed on thin film arrays, fixed targets or using an LCP extruder and several methods including light activation, rapid mixing and the use of photocages have effectively been used to initiate reactions in micro-crystalline slurries for dynamic studies.
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Recent developments on the beamline will be presented including optimisation of fixed-targets for time-resolved SSX challenging targets requiring anaerobic or dark conditions, exploring the advantages of high energy data collection and sophisticated new detectors, fast grid scans, multi-crystal data collection and complementary spectroscopic methods.
Structural and biochemical basis of USP1 inhibition by small molecules
Dr Martin Rennie, University of Glasgow
Ubiquitin-specific protease 1 (USP1) is involved in DNA repair and a potential target for treatment of cancers with homologous recombination deficiencies. This enzyme deubiquitinates DNA clamps that are site-specifically mono-ubiquitinated. (De-)mono-ubiquitination acts to regulate the protein-protein and protein-DNA interactions of the clamps, thereby controlling the functions of the clamps in DNA repair.
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Several small molecule inhibitors of USP1 are currently in phase I clinical trials, alone and in combination with PARP inhibitors, for the treatment of certain tumours. We used cryo-electron microscopy and biochemical assays to probe the mechanism of action of two USP1 inhibitors, ML323 and KSQ-4279. We found that both inhibitors bind to a cryptic site of USP1 and allosterically disrupt the active site. Inhibitor binding drove a substantial increase in thermal stability of USP1, which may be due to the filling of a hydrophobic channel in the enzyme. Our results uncover the mechanism of action of USP1 inhibitors at the molecular level.
Post-translational modifications in the Protein Data Bank
Lucy Schofield, University of York
Proteins frequently undergo covalent modification at the post-translational level, a process that involves the covalent attachment of various chemical groups to specific amino acids within a protein. These modifications are known as post-translational modifications (PTMs) and range from the simple addition of small functional groups, as observed in phosphorylation, to more complex alterations such as glycosylation, where carbohydrate moieties are added. Additionally, some modifications involve the transformation of amino acids, for example the formation of pyroglutamic acid. These post-translational modifications (PTMs) are essential for the normal functioning of cells, as they can significantly alter the physicochemical properties of amino acids. Consequently, PTMs influence crucial biological processes, including enzymatic activity, protein localization, protein–protein interactions, and overall protein stability.
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Identifying PTMs within the Protein Data Bank (PDB) is complex, as there is currently no single data item in mmCIF files that facilitates the search for structures containing PTMs, making comprehensive analysis difficult. In this talk, I will present the analysis of PTM prevalence in the PDB, which was performed through a substructure search across multiple databases. We will examine the occurrence and distribution of various types of PTMs within the PDB and discuss their biological implications. Despite the critical roles that PTMs play in cellular function and regulation, accurately representing them in three-dimensional protein structures remains a significant challenge. I will therefore present examples of accurately modelled PTMs, which can serve as valuable references for future modelling efforts.