ORCID Profile
0000-0003-0378-0017
Current Organisations
Diamond Light Source
,
University of Oxford
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Publisher: MDPI AG
Date: 19-09-2018
DOI: 10.20944/PREPRINTS201809.0383.V1
Abstract: The XChem facility at Diamond Light Source offers fragment screening by X-ray crystallography as a general access user program. & nbsp The main advantage of X-ray crystallography as a primary fragment screen is that it yields directly the location and pose of the fragment hits, whether within pockets of interest or merely on surface sites:& nbsp this is the key information for structure-based design and for enabling synthesis of follow-up molecules. Extensive streamlining of the screening experiment at XChem has engendered a very active user programme that is generating large amounts of data: & nbsp in 2017, 36 academic and industry groups generated 35,000 datasets of uniquely soaked crystals. & nbsp It has also generated a large number of learnings concerning the main remaining bottleneck, namely obtaining a suitable crystal system that will support a successful fragment screen.& nbsp Here we discuss the practicalities of generating screen-ready crystals that have useful electron density maps, and how to ensure they will be successfully reproduced and usable at a facility outside the home lab.
Publisher: International Union of Crystallography (IUCr)
Date: 2021
DOI: 10.1107/S2059798320014114
Abstract: Despite the tremendous success of X-ray cryo-crystallography in recent decades, the transfer of crystals from the drops in which they are grown to diffractometer s le mounts remains a manual process in almost all laboratories. Here, the Shifter, a motorized, interactive microscope stage that transforms the entire crystal-mounting workflow from a rate-limiting manual activity to a controllable, high-throughput semi-automated process, is described. By combining the visual acuity and fine motor skills of humans with targeted hardware and software automation, it was possible to transform the speed and robustness of crystal mounting. Control software, triggered by the operator, manoeuvres crystallization plates beneath a clear protective cover, allowing the complete removal of film seals and thereby eliminating the tedium of repetitive seal cutting. The software, either upon request or working from an imported list, controls motors to position crystal drops under a hole in the cover for human mounting at a microscope. The software automatically captures experimental annotations for uploading to the user's data repository, removing the need for manual documentation. The Shifter facilitates mounting rates of 100–240 crystals per hour in a more controlled process than manual mounting, which greatly extends the lifetime of the drops and thus allows a dramatic increase in the number of crystals retrievable from any given drop without loss of X-ray diffraction quality. In 2015, the first in a series of three Shifter devices was deployed as part of the XChem fragment-screening facility at Diamond Light Source, where they have since facilitated the mounting of over 120 000 crystals. The Shifter was engineered to have a simple design, providing a device that could be readily commercialized and widely adopted owing to its low cost. The versatile hardware design allows use beyond fragment screening and protein crystallography.
Publisher: Cold Spring Harbor Laboratory
Date: 05-03-2018
DOI: 10.1101/073411
Abstract: Macromolecular crystallography is relied on to reveal subtle atomic difference between s les (e.g. ligand binding) yet their detection and modelling is subjective and ambiguous density is experimentally common, since molecular states of interest are generally only fractionally present. The existing approach relies on careful modelling for maximally accurate maps to make contributions of the minor fractions visible ( 1 ) in practice, this is time-consuming and non-objective ( 2 – 4 ). Instead, our PanDDA method automatically reveals clear electron density for only the changed state, even from poor models and inaccurate maps, by subtracting a proportion of the confounding ground state, accurately estimated by averaging many ground state crystals. Changed states are objectively identifiable from statistical distributions of density values arbitrarily large searches are thus automatable. The method is completely general, implying new best practice for all changed-state studies. Finally, we demonstrate the incompleteness of current atomic models, and the need for new multi-crystal deconvolution paradigms. Normally uninterpretable map regions are reliably modelled by deconvoluting superposed crystal states, even with poor starting models.
Publisher: American Chemical Society (ACS)
Date: 11-07-2012
DOI: 10.1021/JM300677J
Publisher: Springer Science and Business Media LLC
Date: 02-09-2020
DOI: 10.1038/S42004-020-00367-0
Abstract: Fragment based methods are now widely used to identify starting points in drug discovery and generation of tools for chemical biology. A significant challenge is optimization of these weak binding fragments to hit and lead compounds. We have developed an approach where in idual reaction mixtures of analogues of hits can be evaluated without purification of the product. Here, we describe experiments to optimise the processes and then assess such mixtures in the high throughput crystal structure determination facility, XChem. Diffraction data for crystals of the proteins Hsp90 and PDHK2 soaked in idually with 83 crude reaction mixtures are analysed manually or with the automated XChem procedures. The results of structural analysis are compared with binding measurements from other biophysical techniques. This approach can transform early hit to lead optimisation and the lessons learnt from this study provide a protocol that can be used by the community.
Publisher: International Union of Crystallography (IUCr)
Date: 27-09-2014
DOI: 10.1107/S1399004714017581
Abstract: The visual inspection of crystallization experiments is an important yet time-consuming and subjective step in X-ray crystallography. Previously published studies have focused on automatically classifying crystallization droplets into distinct but ultimately arbitrary experiment outcomes here, a method is described that instead ranks droplets by their likelihood of containing crystals or microcrystals, thereby prioritizing for visual inspection those images that are most likely to contain useful information. The use of textons is introduced to describe crystallization droplets objectively, allowing them to be scored with the posterior probability of a random forest classifier trained against droplets manually annotated for the presence or absence of crystals or microcrystals. Unlike multi-class classification, this two-class system lends itself naturally to unidirectional ranking, which is most useful for assisting sequential viewing because images can be arranged simply by using these scores: this places droplets with probable crystalline behaviour early in the viewing order. Using this approach, the top ten wells included at least one human-annotated crystal or microcrystal for 94% of the plates in a data set of 196 plates imaged with a Minstrel HT system. The algorithm is robustly transferable to at least one other imaging system: when the parameters trained from Minstrel HT images are applied to a data set imaged by the Rock Imager system, human-annotated crystals ranked in the top ten wells for 90% of the plates. Because rearranging images is fundamental to the approach, a custom viewer was written to seamlessly support such ranked viewing, along with another important output of the algorithm, namely the shape of the curve of scores, which is itself a useful overview of the behaviour of the plate additional features with known usefulness were adopted from existing viewers. Evidence is presented that such ranked viewing of images allows faster but more accurate evaluation of drops, in particular for the identification of microcrystals.
Publisher: Elsevier BV
Date: 12-2010
DOI: 10.1016/J.CHEMBIOL.2010.09.016
Abstract: The final step in carnitine biosynthesis is catalyzed by γ-butyrobetaine (γBB) hydroxylase (BBOX), an iron/2-oxoglutarate (2OG) dependent oxygenase. BBOX is inhibited by trimethylhydrazine-propionate (THP), a clinically used compound. We report structural and mechanistic studies on BBOX and its reaction with THP. Crystallographic and sequence analyses reveal that BBOX and trimethyllysine hydroxylase form a subfamily of 2OG oxygenases that dimerize using an N-terminal domain. The crystal structure reveals the active site is enclosed and how THP competes with γBB. THP is a substrate giving formaldehyde (supporting structural links with histone demethylases), dimethylamine, malonic acid semi-aldehyde, and an unexpected product with an additional carbon-carbon bond resulting from N-demethylation coupled to oxidative rearrangement, likely via an unusual radical mechanism. The results provide a basis for development of improved BBOX inhibitors and may inspire the discovery of additional rearrangement reactions.
Publisher: American Chemical Society (ACS)
Date: 27-10-2017
DOI: 10.1021/ACS.JMEDCHEM.7B00933
Abstract: Inhibitors of HDAC6 have attractive potential in numerous cancers. HDAC6 inhibitors to date target the catalytic domains, but targeting the unique zinc-finger ubiquitin-binding domain (Zf-UBD) of HDAC6 may be an attractive alternative strategy. We developed X-ray crystallography and biophysical assays to identify and characterize small molecules capable of binding to the Zf-UBD and competing with ubiquitin binding. Our results revealed two adjacent ligand-able pockets of HDAC6 Zf-UBD and the first functional ligands for this domain.
Publisher: Cold Spring Harbor Laboratory
Date: 26-07-2022
DOI: 10.1101/2022.07.26.501559
Abstract: Obtaining protein crystals suitable for characterizing ligands and understanding different protein conformations remains a bottleneck for structural studies. Using nanobodies as crystallization chaperones is one strategy to address the problem, but its reliability is uncharacterized and in this study, we observed it to have a poor success rate. We therefore set out to engineer robust crystallization behaviour into the nanobody scaffold by exploring the nanobody-nanobody interface predominant in the PDB using combinations of surface mutations. This yielded multiple polymorphs, all mediated by this interface, with one providing far superior resolution and reliability of diffraction. We therefore refer to the modified nanobody scaffolds as “Gluebodies” and propose at least 2 variants that should be routinely generated for chaperone experiments. We furthermore show that Gluedodies cannot rescue intrinsically non-crystallizing proteins, but are a powerful approach to improve the packing and resolution limit of poorly diffracting crystals.
Publisher: Elsevier
Date: 2018
DOI: 10.1016/BS.MIE.2018.09.027
Abstract: The XChem facility at Diamond Light Source offers fragment screening by X-ray crystallography as a general access user program. The main advantage of X-ray crystallography as a primary fragment screen is that it yields directly the location and pose of the fragment hits, whether within pockets of interest or merely on surface sites: this is the key information for structure-based design and for enabling synthesis of follow-up molecules. Extensive streamlining of the screening experiment at XChem has engendered a very active user program that is generating large amounts of data: in 2017, 36 academic and industry groups generated 35,000 datasets of uniquely soaked crystals. It has also generated a large number of learnings concerning the main remaining bottleneck, namely, obtaining a suitable crystal system that will support a successful fragment screen. Here we discuss the practicalities of generating screen-ready crystals that have useful electron density maps, and how to ensure they will be successfully reproduced and usable at a facility outside the home lab.
Publisher: Wiley
Date: 26-04-2019
Abstract: Historically, chemists have explored chemical space in a highly uneven and unsystematic manner. As an ex le, the shape ersity of existing fragment sets does not generally reflect that of all theoretically possible fragments. To assess experimentally the added value of increased three dimensionality, a shape- erse fragment set was designed and collated. The set was assembled by both using commercially available fragments and harnessing unified synthetic approaches to sp
Publisher: International Union of Crystallography (IUCr)
Date: 24-02-2017
DOI: 10.1107/S2059798316020234
Abstract: XChemExplorer ( XCE ) is a data-management and workflow tool to support large-scale simultaneous analysis of protein–ligand complexes during structure-based ligand discovery (SBLD). The user interfaces of established crystallographic software packages such as CCP 4 [Winn et al. (2011), Acta Cryst. D 67 , 235–242] or PHENIX [Adams et al. (2010), Acta Cryst. D 66 , 213–221] have entrenched the paradigm that a `project' is concerned with solving one structure. This does not hold for SBLD, where many almost identical structures need to be solved and analysed quickly in one batch of work. Functionality to track progress and annotate structures is essential. XCE provides an intuitive graphical user interface which guides the user from data processing, initial map calculation, ligand identification and refinement up until data dissemination. It provides multiple entry points depending on the need of each project, enables batch processing of multiple data sets and records metadata, progress and annotations in an SQLite database. XCE is freely available and works on any Linux and Mac OS X system, and the only dependency is to have the latest version of CCP 4 installed. The design and usage of this tool are described here, and its usefulness is demonstrated in the context of fragment-screening c aigns at the Diamond Light Source. It is routinely used to analyse projects comprising 1000 data sets or more, and therefore scales well to even very large ligand-design projects.
Publisher: The Royal Society
Date: 29-04-2019
Abstract: Structure-guided drug discovery emerged in the 1970s and 1980s, stimulated by the three-dimensional structures of protein targets that became available, mainly through X-ray crystal structure analysis, assisted by the development of synchrotron radiation sources. Structures of known drugs or inhibitors were used to guide the development of leads. The growth of high-throughput screening during the late 1980s and the early 1990s in the pharmaceutical industry of chemical libraries of hundreds of thousands of compounds of molecular weight of approximately 500 Da was impressive but still explored only a tiny fraction of the chemical space of the predicted 10 40 drug-like compounds. The use of fragments with molecular weights less than 300 Da in drug discovery not only decreased the chemical space needing exploration but also increased promiscuity in binding targets. Here we discuss advances in X-ray fragment screening and the challenge of identifying sites where fragments not only bind but can be chemically elaborated while retaining their positions and binding modes. We first describe the analysis of fragment binding using conventional X-ray difference Fourier techniques, with Mycobacterium abscessus SAICAR synthetase (PurC) as an ex le. We observe that all fragments occupy positions predicted by computational hotspot mapping. We compare this with fragment screening at Diamond Synchrotron Light Source XChem facility using PanDDA software, which identifies many more fragment hits, only some of which bind to the predicted hotspots. Many low occupancy sites identified may not support elaboration to give adequate ligand affinity, although they will likely be useful in drug discovery as ‘warm spots’ for guiding elaboration of fragments bound at hotspots. We discuss implications of these observations for fragment screening at the synchrotron sources. This article is part of the theme issue ‘Fifty years of synchrotron science: achievements and opportunities’.
Publisher: Elsevier BV
Date: 07-2018
Publisher: Springer Science and Business Media LLC
Date: 24-04-2017
DOI: 10.1038/NCOMMS15123
Abstract: In macromolecular crystallography, the rigorous detection of changed states (for ex le, ligand binding) is difficult unless signal is strong. Ambiguous (‘weak’ or ‘noisy’) density is experimentally common, since molecular states are generally only fractionally present in the crystal. Existing methodologies focus on generating maximally accurate maps whereby minor states become discernible in practice, such map interpretation is disappointingly subjective, time-consuming and methodologically unsound. Here we report the PanDDA method, which automatically reveals clear electron density for the changed state—even from inaccurate maps—by subtracting a proportion of the confounding ‘ground state’ changed states are objectively identified from statistical analysis of density distributions. The method is completely general, implying new best practice for all changed-state studies, including the routine collection of multiple ground-state crystals. More generally, these results demonstrate: the incompleteness of atomic models that single data sets contain insufficient information to model them fully and that accuracy requires further map-deconvolution approaches.
Publisher: F1000 Research Ltd
Date: 11-06-2021
DOI: 10.12688/WELLCOMEOPENRES.16847.1
Abstract: There is an increasingly urgent need for new antibiotics, yet there is a significant and persistent economic problem when it comes to developing such medicines. The problem stems from the perceived need for a “market” to drive commercial antibiotic development. In this article, we explore abandoning the market as a prerequisite for successful antibiotic research and development. Once one stops trying to fix a market model that has stopped functioning, one is free to carry out research and development (R& D) in ways that are more openly collaborative, a mechanism that has been demonstrably effective for the R& D underpinning the response to the COVID pandemic. New “open source” research models have great potential for the development of medicines for areas of public health where the traditional profit-driven model struggles to deliver. New financial initiatives, including major push ull incentives, aimed at fixing the broken antibiotics market provide one possible means for funding an openly collaborative approach to drug development. We argue that now is therefore the time to evaluate, at scale, whether such methods can deliver new medicines through to patients, in a timely manner.
Publisher: Proceedings of the National Academy of Sciences
Date: 23-03-2015
Abstract: RecQ DNA helicases are critical enzymes for the maintenance of genome integrity. Here, we determined the first DNA complex structures, to our knowledge, of the human RECQ1 helicase. These structures provide new insight into the RecQ helicase mechanism of DNA tracking, strand separation, strand annealing, and Holliday junction (HJ) branch migration. We identified a surface region in the winged-helix domain of RECQ1 that is important for both dsDNA recognition and HJ resolution, and we used a combination of biochemical, analytical ultracentrifugation, and EM experiments to begin elucidating the molecular basis of the distinct HJ resolution activities of human RecQ helicases.
Publisher: Wiley
Date: 06-10-2017
Publisher: Proceedings of the National Academy of Sciences
Date: 20-04-2011
Abstract: Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X) 18 -E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528 R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.
Publisher: Cold Spring Harbor Laboratory
Date: 30-10-2020
DOI: 10.1101/2020.10.29.339317
Abstract: The COVID-19 pandemic was a stark reminder that a barren global antiviral pipeline has grave humanitarian consequences. Pandemics could be prevented in principle by accessible, easily deployable broad-spectrum oral antivirals. Here we report the results of the COVID Moonshot , a fully open-science, crowd sourced, structure-enabled drug discovery c aign targeting the SARS-CoV-2 main protease. We discovered a novel chemical series that is differentiated from current Mpro inhibitors in that it maintains a new non-covalent, non-peptidic scaffold with nanomolar potency. Our approach leveraged crowdsourcing, high-throughput structural biology, machine learning, and exascale molecular simulations and high-throughput chemistry. In the process, we generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. In a first for a structure-based drug discovery c aign, all compound designs ( ,000 designs), crystallographic data ( ligand-bound X-ray structures), assay data ( ,000 measurements), and synthesized molecules ( ,400 compounds) for this c aign were shared rapidly and openly, creating a rich open and IP-free knowledgebase for future anti-coronavirus drug discovery.
Publisher: International Union of Crystallography (IUCr)
Date: 03-2017
DOI: 10.1107/S205979831700331X
Abstract: The steady expansion in the capacity of modern beamlines for high-throughput data collection, enabled by increasing X-ray brightness, capacity of robotics and detector speeds, has pushed the bottleneck upstream towards s le preparation. Even in ligand-binding studies using crystal soaking, the experiment best able to exploit beamline capacity, a primary limitation is the need for gentle and nontrivial soaking regimens such as stepwise concentration increases, even for robust and well characterized crystals. Here, the use of acoustic droplet ejection for the soaking of protein crystals with small molecules is described, and it is shown that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 min. In addition to having very low compound consumption (tens of nanolitres per s le), the positional precision of acoustic droplet ejection enables the targeted placement of the compound/solvent away from crystals and towards drop edges, allowing gradual diffusion of solvent across the drop. This ensures both an improvement in the reproducibility of X-ray diffraction and increased solvent tolerance of the crystals, thus enabling higher effective compound-soaking concentrations. The technique is detailed here with ex les from the protein target JMJD2D, a histone lysine demethylase with roles in cancer and the focus of active structure-based drug-design efforts.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5SC03115J
Abstract: High concentration crystal soaking of poised fragments and one-step elaboration identified compound 17 as an inhibitor of the PHIP(2) bromodomain.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Frank von Delft.