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Open Access
Abstract: DNA gyrase is a bacterial type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology and an archetypical target of antibiotics. The widely used quinolone class of drugs use a water–metal ion bridge in interacting with the GyrA subunit of DNA gyrase. Zoliflodacin sits in the same pocket as quinolones but interacts with the GyrB subunit and also stabilizes lethal double-stranded DNA breaks. Gepotidacin has been observed to sit on the twofold axis of the complex, midway between the two four-base-pair separated DNA-cleavage sites and has been observed to stabilize singe-stranded DNA breaks. Here, we use information from three crystal structures of complexes of Staphlococcus aureus DNA gyrase (one with a precursor of gepotidacin and one with the progenitor of zoliflodacin) to propose a simple single moving metal-ion-catalyzed DNA-cleavage mechanism. Our model explains why the catalytic tyrosine is in the tyrosinate (negatively charged) form for DNA cleavage. Movement of a single catalytic metal-ion (Mg2+ or Mn2+) guides water-mediated protonation and cleavage of the scissile phosphate, which is then accepted by the catalytic tyrosinate. Type IIA topoisomerases need to be able to rapidly cut the DNA when it becomes positively supercoiled (in front of replication forks and transcription bubbles) and we propose that the original purpose of the small Greek Key domain, common to all type IIA topoisomerases, was to allow access of the catalytic metal to the DNA-cleavage site. Although the proposed mechanism is consistent with published data, it is not proven and other mechanisms have been proposed. Finally, how such mechanisms can be experimentally distinguished is considered.
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Dec 2024
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I23-Long wavelength MX
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Diamond Proposal Number(s):
[29990]
Open Access
Abstract: One of the challenges for experimental structural biology in the 21st century is to see chemical reactions happen. Staphylococcus aureus (S. aureus) DNA gyrase is a type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology. Drugs, such as gepotidacin, zoliflodacin and the quinolone moxifloxacin, can stabilize these normally transient DNA strand breaks and kill bacteria. Crystal structures of uncleaved DNA with a gepotidacin precursor (2.1 Å GSK2999423) or with doubly cleaved DNA and zoliflodacin (or with its progenitor QPT-1) have been solved in the same P61 space-group (a = b ≈ 93 Å, c ≈ 412 Å). This suggests that it may be possible to observe the two DNA cleavage steps (and two DNA-religation steps) in this P61 space-group. Here, a 2.58 Å anomalous manganese dataset in this crystal form is solved, and four previous crystal structures (1.98 Å, 2.1 Å, 2.5 Å and 2.65 Å) in this crystal form are re-refined to clarify crystal contacts. The structures clearly suggest a single moving metal mechanism—presented in an accompanying (second) paper. A previously published 2.98 Å structure of a yeast topoisomerase II, which has static disorder around a crystallographic twofold axis, was published as containing two metals at one active site. Re-refined coordinates of this 2.98 Å yeast structure are consistent with other type IIA topoisomerase structures in only having one metal ion at each of the two different active sites.
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Nov 2024
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Catherine L.
Lawson
,
Andriy
Kryshtafovych
,
Grigore D.
Pintilie
,
Stephen
Burley
,
Jiří
Černý
,
Vincent B.
Chen
,
Paul
Emsley
,
Alberto
Gobbi
,
Andrzej
Joachimiak
,
Sigrid
Noreng
,
Michael G.
Prisant
,
Randy J.
Read
,
Jane S.
Richardson
,
Alexis L.
Rohou
,
Bohdan
Schneider
,
Benjamin D.
Sellers
,
Chenghua
Shao
,
Elizabeth
Sourial
,
Chris I.
Williams
,
Christopher J.
Williams
,
Ying
Yang
,
Venkat
Abbaraju
,
Pavel V.
Afonine
,
Matthew L.
Baker
,
Paul S.
Bond
,
Tom L.
Blundell
,
Tom
Burnley
,
Arthur
Campbell
,
Renzhi
Cao
,
Jianlin
Cheng
,
Grzegorz
Chojnowski
,
Kevin D.
Cowtan
,
Frank
Dimaio
,
Reza
Esmaeeli
,
Nabin
Giri
,
Helmut
Grubmüller
,
Soon Wen
Hoh
,
Jie
Hou
,
Corey F.
Hryc
,
Carola
Hunte
,
Maxim
Igaev
,
Agnel P.
Joseph
,
Wei-Chun
Kao
,
Daisuke
Kihara
,
Dilip
Kumar
,
Lijun
Lang
,
Sean
Lin
,
Sai R.
Maddhuri Venkata Subramaniya
,
Sumit
Mittal
,
Arup
Mondal
,
Nigel W.
Moriarty
,
Andrew
Muenks
,
Garib N.
Murshudov
,
Robert A.
Nicholls
,
Mateusz
Olek
,
Colin M.
Palmer
,
Alberto
Perez
,
Emmi
Pohjolainen
,
Karunakar R.
Pothula
,
Christopher N.
Rowley
,
Daipayan
Sarkar
,
Luisa U.
Schäfer
,
Christopher J.
Schlicksup
,
Gunnar F.
Schröder
,
Mrinal
Shekhar
,
Dong
Si
,
Abhishek
Singharoy
,
Oleg V.
Sobolev
,
Genki
Terashi
,
Andrea C.
Vaiana
,
Sundeep C.
Vedithi
,
Jacob
Verburgt
,
Xiao
Wang
,
Rangana
Warshamanage
,
Martyn
Winn
,
Simone
Weyand
,
Keitaro
Yamashita
,
Minglei
Zhao
,
Michael F.
Schmid
,
Helen M.
Berman
,
Wah
Chiu
Abstract: The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein–nucleic acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9–2.5 Å) resolution. Three published maps were selected as targets: Escherichia coli beta-galactosidase with inhibitor, SARS-CoV-2 virus RNA-dependent RNA polymerase with covalently bound nucleotide analog and SARS-CoV-2 virus ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. The quality of submitted ligand models and surrounding atoms were analyzed by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics and contact scores. A composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.
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Jun 2024
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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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Chloe R.
Koulouris
,
Sian E.
Gardiner
,
Tessa K.
Harris
,
Karen T.
Elvers
,
S. Mark
Roe
,
Jason A.
Gillespie
,
Simon E.
Ward
,
Olivera
Grubisha
,
Robert A.
Nicholls
,
John R.
Atack
,
Benjamin D.
Bax
Diamond Proposal Number(s):
[19990]
Open Access
Abstract: Human serine racemase (hSR) catalyses racemisation of L-serine to D-serine, the latter of which is a co-agonist of the NMDA subtype of glutamate receptors that are important in synaptic plasticity, learning and memory. In a ‘closed’ hSR structure containing the allosteric activator ATP, the inhibitor malonate is enclosed between the large and small domains while ATP is distal to the active site, residing at the dimer interface with the Tyr121 hydroxyl group contacting the α-phosphate of ATP. In contrast, in ‘open’ hSR structures, Tyr121 sits in the core of the small domain with its hydroxyl contacting the key catalytic residue Ser84. The ability to regulate SR activity by flipping Tyr121 from the core of the small domain to the dimer interface appears to have evolved in animals with a CNS. Multiple X-ray crystallographic enzyme-fragment structures show Tyr121 flipped out of its pocket in the core of the small domain. Data suggest that this ligandable pocket could be targeted by molecules that inhibit enzyme activity.
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Apr 2022
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Liz
Potterton
,
Jon
Agirre
,
Charles
Ballard
,
Kevin
Cowtan
,
Eleanor
Dodson
,
Phil R.
Evans
,
Huw T.
Jenkins
,
Ronan
Keegan
,
Eugene
Krissinel
,
Kyle
Stevenson
,
Andrey
Lebedev
,
Stuart J.
Mcnicholas
,
Robert A.
Nicholls
,
Martin
Noble
,
Navraj S.
Pannu
,
Christian
Roth
,
George
Sheldrick
,
Pavol
Skubak
,
Johan
Turkenburg
,
Ville
Uski
,
Frank
Von Delft
,
David
Waterman
,
Keith
Wilson
,
Martyn
Winn
,
Marcin
Wojdyr
Open Access
Abstract: The CCP4 (Collaborative Computational Project, Number 4) software suite for macromolecular structure determination by X-ray crystallography groups brings together many programs and libraries that, by means of well established conventions, interoperate effectively without adhering to strict design guidelines. Because of this inherent flexibility, users are often presented with diverse, even divergent, choices for solving every type of problem. Recently, CCP4 introduced CCP4i2, a modern graphical interface designed to help structural biologists to navigate the process of structure determination, with an emphasis on pipelining and the streamlined presentation of results. In addition, CCP4i2 provides a framework for writing structure-solution scripts that can be built up incrementally to create increasingly automatic procedures.
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Feb 2018
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Data acquisition
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Open Access
Abstract: In this paper, AUSPEX, a new software tool for experimental X-ray data analysis, is presented. Exploring the behaviour of diffraction intensities and the associated estimated uncertainties facilitates the discovery of underlying problems and can help users to improve their data acquisition and processing in order to obtain better structural models. The program enables users to inspect the distribution of observed intensities (or amplitudes) against resolution as well as the associated estimated uncertainties (sigmas). It is demonstrated how AUSPEX can be used to visually and automatically detect ice-ring artefacts in integrated X-ray diffraction data. Such artefacts can hamper structure determination, but may be difficult to identify from the raw diffraction images produced by modern pixel detectors. The analysis suggests that a significant portion of the data sets deposited in the PDB contain ice-ring artefacts. Furthermore, it is demonstrated how other problems in experimental X-ray data caused, for example, by scaling and data-conversion procedures can be detected by AUSPEX.
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Sep 2017
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