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Daren
Fearon
,
Ailsa
Powell
,
Alice
Douangamath
,
Alexandre
Dias
,
Charles W. E.
Tomlinson
,
Blake H.
Balcomb
,
Jasmin C.
Aschenbrenner
,
Anthony
Aimon
,
Isabel A.
Barker
,
Jose
Brandao-Neto
,
Patrick
Collins
,
Louise E.
Dunnett
,
Michael
Fairhead
,
Richard J.
Gildea
,
Mathew
Golding
,
Tyler
Gorrie-Stone
,
Paul V.
Hathaway
,
Lizbe
Koekemoer
,
Tobias
Krojer
,
Ryan
Lithgo
,
Elizabeth M.
Maclean
,
Peter G.
Marples
,
Xiaomin
Ni
,
Rachael
Skyner
,
Romain
Talon
,
Warren
Thompson
,
Conor F.
Wild
,
Max
Winokan
,
Nathan D.
Wright
,
Graeme
Winter
,
Elizabeth J.
Shotton
,
Frank
Von Delft
Open Access
Abstract: Fragment-based drug discovery is a well-established method for the identification of chemical starting points for development into clinical candidates. Historically, crystallographic fragment screening was perceived to be low-throughput and time consuming. However, thanks to advances in synchrotron capabilities and the introduction of dedicated facilities, such as the XChem platform at Diamond Light Source, there have been substantial improvements in throughput and integration between sample preparation, data collection and hit identification. Herein we share our experiences of establishing a crystallographic fragment screening facility, our learnings from operating a user programme for ten years and our perspective on applying structural enablement to rapidly progress initial fragment hits to lead-like molecules.
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Nov 2024
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I04-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Halina
Mikolajek
,
Juan
Sanchez-Weatherby
,
James
Sandy
,
Richard J.
Gildea
,
Ivan
Campeotto
,
Harish
Cheruvara
,
John D.
Clarke
,
Toshana
Foster
,
Sotaro
Fujii
,
Ian T.
Paulsen
,
Bhumika S.
Shah
,
Michael A.
Hough
Open Access
Abstract: The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention.
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Jul 2023
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I24-Microfocus Macromolecular Crystallography
VMXm-Versatile Macromolecular Crystallography microfocus
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Jeremy R.
Keown
,
Adam D.
Crawshaw
,
Jose
Trincao
,
Loic
Carrique
,
Richard J.
Gildea
,
Sam
Horrell
,
Anna J.
Warren
,
Danny
Axford
,
Robin
Owen
,
Gwyndaf
Evans
,
Annie
Bézier
,
Peter
Metcalf
,
Jonathan M.
Grimes
Diamond Proposal Number(s):
[19946, 23570, 27314, 28534]
Open Access
Abstract: Infectious protein crystals are an essential part of the viral lifecycle for double-stranded DNA Baculoviridae and double-stranded RNA cypoviruses. These viral protein crystals, termed occlusion bodies or polyhedra, are dense protein assemblies that form a crystalline array, encasing newly formed virions. Here, using X-ray crystallography we determine the structure of a polyhedrin from Nudiviridae. This double-stranded DNA virus family is a sister-group to the baculoviruses, whose members were thought to lack occlusion bodies. The 70-year-old sample contains a well-ordered lattice formed by a predominantly α-helical building block that assembles into a dense, highly interconnected protein crystal. The lattice is maintained by extensive hydrophobic and electrostatic interactions, disulfide bonds, and domain switching. The resulting lattice is resistant to most environmental stresses. Comparison of this structure to baculovirus or cypovirus polyhedra shows a distinct protein structure, crystal space group, and unit cell dimensions, however, all polyhedra utilise common principles of occlusion body assembly.
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Jul 2023
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Jon
Agirre
,
Mihaela
Atanasova
,
Haroldas
Bagdonas
,
Charles B.
Ballard
,
Arnaud
Basle
,
James
Beilsten-Edmands
,
Rafael J.
Borges
,
David G.
Brown
,
J. Javier
Burgos-Marmol
,
John M.
Berrisford
,
Paul S.
Bond
,
Iracema
Caballero
,
Lucrezia
Catapano
,
Grzegorz
Chojnowski
,
Atlanta G.
Cook
,
Kevin D.
Cowtan
,
Tristan I.
Croll
,
Judit É.
Debreczeni
,
Nicholas E.
Devenish
,
Eleanor J.
Dodson
,
Tarik R.
Drevon
,
Paul
Emsley
,
Gwyndaf
Evans
,
Phil R.
Evans
,
Maria
Fando
,
James
Foadi
,
Luis
Fuentes-Montero
,
Elspeth F.
Garman
,
Markus
Gerstel
,
Richard J.
Gildea
,
Kaushik
Hatti
,
Maarten L.
Hekkelman
,
Philipp
Heuser
,
Soon Wen
Hoh
,
Michael A.
Hough
,
Huw T.
Jenkins
,
Elisabet
Jiménez
,
Robbie P.
Joosten
,
Ronan M.
Keegan
,
Nicholas
Keep
,
Eugene B.
Krissinel
,
Petr
Kolenko
,
Oleg
Kovalevskiy
,
Victor S.
Lamzin
,
David M.
Lawson
,
Andrey
Lebedev
,
Andrew G. W.
Leslie
,
Bernhard
Lohkamp
,
Fei
Long
,
Martin
Maly
,
Airlie
Mccoy
,
Stuart J.
Mcnicholas
,
Ana
Medina
,
Claudia
Millán
,
James W.
Murray
,
Garib N.
Murshudov
,
Robert A.
Nicholls
,
Martin E. M.
Noble
,
Robert
Oeffner
,
Navraj S.
Pannu
,
James M.
Parkhurst
,
Nicholas
Pearce
,
Joana
Pereira
,
Anastassis
Perrakis
,
Harold R.
Powell
,
Randy J.
Read
,
Daniel J.
Rigden
,
William
Rochira
,
Massimo
Sammito
,
Filomeno
Sanchez Rodriguez
,
George M.
Sheldrick
,
Kathryn L.
Shelley
,
Felix
Simkovic
,
Adam J.
Simpkin
,
Pavol
Skubak
,
Egor
Sobolev
,
Roberto A.
Steiner
,
Kyle
Stevenson
,
Ivo
Tews
,
Jens M. H.
Thomas
,
Andrea
Thorn
,
Josep Triviño
Valls
,
Ville
Uski
,
Isabel
Uson
,
Alexei
Vagin
,
Sameer
Velankar
,
Melanie
Vollmar
,
Helen
Walden
,
David
Waterman
,
Keith S.
Wilson
,
Martyn
Winn
,
Graeme
Winter
,
Marcin
Wojdyr
,
Keitaro
Yamashita
Open Access
Abstract: The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.
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Jun 2023
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Open Access
Abstract: Historically, solving the structure of a protein required deep knowledge of crystallography and the ability to produce protein crystals of suitable quality to generate high-quality diffraction data. Over the years, as beamline optics, end-stations, detectors, and data collection strategies have improved, it has become more feasible to extract high-quality diffraction data from ever smaller or less perfect protein crystals and from very large arrays of crystals for techniques such as serial synchrotron crystallography and fragment-based drug discovery. At Diamond, these improvements have been coupled with highly integrated automated pipelines for data reduction and structure solution using techniques such as molecular replacement and experimental phasing. This has led to the dichotomy, and benefits, of being able to do increasingly challenging experiments requiring deep crystallographic knowledge with facility staff support at the same time as lowering the barrier to entry where automated structure solution tools of the facility perform this task for those scientists with less experience. This enables users to focus on the science rather than the process.
Diamond Light Source, the UK’s national synchrotron, has a suite of instruments dedicated to solving the 3D structure of large biological molecules, including seven macromolecular crystallography (MX) beamlines. Solved 3D structures are deposited into the publicly available Protein Data Bank (PDB) and the depositions are released on a weekly basis. In 2020, following 13 years of operation, Diamond hit the milestone of 10,000 structures deposited in the PDB. Two years on, this number is now more than 12,000. Thanks to decades of work across the world, there is an ocean of information in the PDB that serves as an invaluable reference when solving the structures of new proteins.
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Oct 2022
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I24-Microfocus Macromolecular Crystallography
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Richard J.
Gildea
,
James
Beilsten-Edmands
,
Danny
Axford
,
Sam
Horrell
,
Pierre
Aller
,
James
Sandy
,
Juan
Sanchez-Weatherby
,
C. David
Owen
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Robin L.
Owen
,
Martin A.
Walsh
,
Graeme
Winter
Diamond Proposal Number(s):
[26986, 27088]
Open Access
Abstract: In macromolecular crystallography, radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals; for example, small-wedge data collections from micro-crystals, in situ room-temperature data collections and data collection from membrane proteins in lipidic mesophases. Whilst the indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. The identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help to optimize the scaling and merging of multiple data sets, a new program, xia2.multiplex, has been developed which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multi-crystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After the description of a number of use cases, the benefit of xia2.multiplex is demonstrated within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment-screening experiments on the SARS-CoV-2 main protease.
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Jun 2022
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Open Access
Abstract: The DIALS software for the processing of X-ray diffraction data is presented, with an emphasis on how the suite may be used as a toolkit for data processing. The description starts with an overview of the history and intent of the toolkit, usage as an automated system, command-line use, and ultimately how new tools can be written using the API to perform bespoke analysis. Consideration is also made to the application of DIALS to techniques outside of macromolecular X-ray crystallography.
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Nov 2021
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Open Access
Abstract: In processing X-ray diffraction data, the intensities obtained from integration of the diffraction images must be corrected for experimental effects in order to place all intensities on a common scale both within and between data collections. Scaling corrects for effects such as changes in sample illumination, absorption and, to some extent, global radiation damage that cause the measured intensities of symmetry-equivalent observations to differ throughout a data set. This necessarily requires a prior evaluation of the point-group symmetry of the crystal. This paper describes and evaluates the scaling algorithms implemented within the DIALS data-processing package and demonstrates the effectiveness and key features of the implementation on example macromolecular crystallographic rotation data. In particular, the scaling algorithms enable new workflows for the scaling of multi-crystal or multi-sweep data sets, providing the analysis required to support current trends towards collecting data from ever-smaller samples. In addition, the implementation of a free-set validation method is discussed, which allows the quantification of the suitability of scaling-model and algorithm choices.
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Apr 2020
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Data acquisition
Detectors
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Open Access
Abstract: The Diamond Light Source data analysis infrastructure, Zocalo, is built on a messaging framework. Analysis tasks are processed by a scalable pool of workers running on cluster nodes. Results can be written to a common file system, sent to another worker for further downstream processing and/or streamed to a LIMS. Zocalo allows increased parallelization of computationally expensive tasks and makes the use of computational resources more efficient. The infrastructure is low-latency, fault-tolerant, and allows for highly dynamic data processing. Moving away from static workflows expressed in shell scripts we can easily re-trigger processing tasks in the event that an issue is found. It allows users to re-run tasks with additional input and ensures that automatically and manually triggered processing results are treated equally. Zocalo was originally conceived to cope with the additional demand on infrastructure by the introduction of Eiger detectors with up to 18 Mpixels and running at up to 560 Hz framerate on single crystal diffraction beamlines. We are now adapting Zocalo to manage processing tasks for ptychography, tomography, cryo-EM, and serial crystallography workloads.
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Oct 2019
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Graeme
Winter
,
Richard J.
Gildea
,
Neil G.
Paterson
,
John
Beale
,
Markus
Gerstel
,
Danny
Axford
,
Melanie
Vollmar
,
Katherine E.
Mcauley
,
Robin L.
Owen
,
Ralf
Flaig
,
Alun W.
Ashton
,
David
Hall
Open Access
Abstract: 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.
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Mar 2019
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