I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure. Surprisingly, proline residues that are highly conserved in class A β-lactamases were found to tolerate various substitutions without large losses in enzyme activity. We investigated the roles of three conserved prolines at positions 107, 226, and 258 in the β-lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time. For the variant Pro107Thr, the crystal structure shows dimer formation resembling domain swapping. It is concluded that the proline substitutions loosen the structure, enhancing multimerization. Even though the enzyme does not lose its properties without the conserved proline residues, the prolines ensure the long-term structural integrity of the enzyme.
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Apr 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-Macromolecular Crystallography
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Ennio A.
D’amico
,
Misbha
Ud Din Ahmad
,
Verena
Cmentowski
,
Mathias
Girbig
,
Franziska
Müller
,
Sabine
Wohlgemuth
,
Andreas
Brockmeyer
,
Stefano
Maffini
,
Petra
Janning
,
Ingrid R.
Vetter
,
Andrew P.
Carter
,
Anastassis
Perrakis
,
Andrea
Musacchio
Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end–directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein–Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein–Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein–Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.
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Nov 2022
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I03-Macromolecular Crystallography
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Leila T.
Alexander
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Rosalba
Lepore
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Andriy
Kryshtafovych
,
Athanasios
Adamopoulos
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Markus
Alahuhta
,
Ann M.
Arvin
,
Yannick J.
Bomble
,
Bettina
Böttcher
,
Cécile
Breyton
,
Valerio
Chiarini
,
Naga Babu
Chinnam
,
Wah
Chiu
,
Krzysztof
Fidelis
,
Rhys
Grinter
,
Gagan D.
Gupta
,
Marcus D.
Hartmann
,
Christopher S.
Hayes
,
Tatjana
Heidebrecht
,
Andrea
Ilari
,
Andrzej
Joachimiak
,
Youngchang
Kim
,
Romain
Linares
,
Andrew L.
Lovering
,
Vladimir V.
Lunin
,
Andrei N.
Lupas
,
Cihan
Makbul
,
Karolina
Michalska
,
John
Moult
,
Prasun K.
Mukherjee
,
William
Nutt
,
Stefan L.
Oliver
,
Anastassis
Perrakis
,
Lucy
Stols
,
John A.
Tainer
,
Maya
Topf
,
Susan E.
Tsutakawa
,
Mauricio
Valdivia‐delgado
,
Torsten
Schwede
Open Access
Abstract: The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.
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Dec 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19800]
Open Access
Abstract: The current rise of antibiotic resistant forms of Mycobacterium tuberculosis is a global health threat that calls for new antibiotics. The β-lactamase BlaC of this pathogen prevents the use of β-lactam antibiotics, except in combination with a β-lactamase inhibitor. To understand if exposure to such inhibitors can easily result in resistance, a BlaC evolution experiment was performed, studying the evolutionary adaptability against the inhibitor sulbactam. Several amino acid substitutions in BlaC were shown to confer reduced sensitivity to sulbactam. The G132S mutation causes a reduction in the rate of nitrocefin and ampicillin hydrolysis and simultaneously reduces the sensitivity for sulbactam inhibition. Introduction of the side chain moiety of Ser132 causes the 104–105 peptide bond to assume the cis conformation and the side chain of Ser104 to be rotated toward the sulbactam adduct with which it forms a hydrogen bond not present in the wild-type enzyme. The gatekeeper residue Ile105 also moves. These changes in the entrance of the active site can explain the decreased affinity of G132S BlaC for both substrates and sulbactam. Our results show that BlaC can easily acquire a reduced sensitivity for sulbactam, with a single-amino acid mutation, which could hinder the use of combination therapies.
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Jul 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[19800]
Open Access
Abstract: The β-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer most inhibitor resistance, K234R, as well as variant G132N that was found previously, were characterized using X-ray crystallography and NMR relaxation experiments to probe structural and dynamic properties. The G132N mutant exists in solution in two, almost equally populated conformations that exchange with a rate of ca. 88 s−1. The conformational change affects a broad region of the enzyme. The crystal structure reveals that the Asn132 side chain forces the peptide bond between Ser104 and Ile105 in a cis-conformation. The crystal structure suggests multiple conformations for several side chains (e.g. Ser104, Ser130) and a short loop (214-216). In the K234R mutant, the active site dynamics are significantly diminished with respect to the wild type enzyme. These results show that multiple evolutionary routes are available to increase inhibitor resistance in BlaC and that active site dynamics on the millisecond time scale are not required for catalytic function.
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May 2021
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Open Access
Abstract: It is about a decade since SPINE – Structural Proteomics IN Europe – was envisaged, set-up and submitted as an idea to the European Commission fifth framework programme. Our concepts were built on the paradigm of the USA-based structural genomics projects (http://www.nigms.nih.gov/psi), the Japanese initiatives (http://www.rsgi.riken.go.jp/rsgi_e), and some national efforts within Europe. At the same time, benefiting from the experience and technology development of the preceding projects, SPINE took an approach different from that of the mainstream of structural genomics: a major success of SPINE was to bring the cutting-edge high-throughput (HTP) technologies to biomedically relevant targets, not only facilitating the determination of no less than 308 novel structures, but also achieving a highly collaborative spirit and a culture of exchange of knowledge in technology. SPINE-2 Complexes (S2C) was both a continuation of SPINE and a whole new venture in its own right. Having largely achieved the integration of HTP technologies at partner sites, S2C aimed to facilitate the study of macromolecular complexes in key areas of biological research (ubiquitin (de-)conjugation, cell cycle and apoptosis, synaptogenesis and neuronal signaling, kinases and phosphatases, transcription receptors and regulation, innate and acquired immunity, and mechanisms in viral infection). S2C also worked to advance ‘wet-lab’ technologies relevant to the study of challenging targets (e.g., production of multi-subunit complexes with an emphasis on eukaryotic systems, biophysical methods to characterize complexes, and library tools for HTP screening), and ‘dry-lab’ technologies (e.g., microcrystal handling, docking software, as well as data management, analysis and dissemination tools). This ambitious agenda was backed-up by the long standing interests of the participant labs in the corresponding research themes, and was supported by a shared enthusiasm for consolidation of technologies and conceptual approaches, which together allowed us to showcase the structural study of complex systems.
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May 2011
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Autotaxin (ATX or ENPP2) is a secreted glycosylated mammalian enzyme that exhibits lysophospholipase D activity, hydrolyzing lysophosphatidylcholine to the signalling lipid lysophosphatidic acid. ATX is an ~100 kDa multi-domain protein encompassing two N-terminal somatomedin B-like domains, a central catalytic phosphodiesterase domain and a C-terminal nuclease-like domain. Protocols for the efficient expression of ATX from stably transfected mammalian HEK293 cells in amounts sufficient for crystallographic studies are reported. Purification resulted in protein that crystallized readily, but various attempts to grow crystals suitable in size for routine crystallographic structure determination were not successful. However, the available micrometre-thick plates diffracted X-rays beyond 2.0 Å resolution and allowed the collection of complete diffraction data to about 2.6 Å resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed.
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Sep 2010
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