I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Hector
Newman
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Alen
Krajnc
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Domenico
Bellini
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Charles J.
Eyermann
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Grant A.
Boyle
,
Neil
Paterson
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Katherine E.
Mcauley
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Robert
Lesniak
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Mukesh
Gangar
,
Frank
Von Delft
,
Jurgen
Brem
,
Kelly
Chibale
,
Christopher J.
Schofield
,
Christopher G.
Dowson
Diamond Proposal Number(s):
[17884]
Open Access
Abstract: The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boron-containing inhibitors are potent serine-β-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) β-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by β-lactamase-driven resistance.
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Jul 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24717]
Open Access
Abstract: This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.
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May 2021
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I03-Macromolecular Crystallography
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Laszlo L. P.
Hosszu
,
Rebecca
Conners
,
Daljit
Sangar
,
Mark
Batchelor
,
Elizabeth B.
Sawyer
,
Stuart
Fisher
,
Matthew J.
Cliff
,
Andrea M.
Hounslow
,
Katherine
Mcauley
,
R. Leo
Brady
,
Graham S.
Jackson
,
Jan
Bieschke
,
Jonathan P.
Waltho
,
John
Collinge
Diamond Proposal Number(s):
[4923, 5969]
Open Access
Abstract: Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease.
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Jul 2020
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B21-High Throughput SAXS
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Open Access
Abstract: It is common to switch between H2O and D2O when examining peptide-based systems with the assumption being that there are no effects from this change. Here, we describe the effect of changing from H2O to D2O in a number of low molecular weight dipeptide-based gels. Gels are formed by decreasing the pH. In most cases, there is little dif-ference in the structures formed at high pH, but this is not universally true. On lowering the pH, the ki-netics of gelation are affected and, in some cases, the structures underpinning the gel network are dif-ferent. Where there are differences in the self-assembled structures, the resulting gel properties are different. We therefore show isotopic control over gel properties is possible.
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Jul 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Sarah L.
Kidd
,
Elaine
Fowler
,
Till
Reinhardt
,
Thomas
Compton
,
Natalia
Mateu
,
Hector
Newman
,
Dom
Bellini
,
Romain
Talon
,
Joseph
Mcloughlin
,
Tobias
Krojer
,
Anthony
Aimon
,
Anthony
Bradley
,
Michael
Fairhead
,
Paul
Brear
,
Laura
Diaz-Saez
,
Katherine
Mcauley
,
Hannah F.
Sore
,
Andrew
Madin
,
Daniel H.
O'Donovan
,
Kilian
Huber
,
Marko
Hyvonen
,
Frank
Von Delft
,
Christopher G.
Dowson
,
David R.
Spring
Diamond Proposal Number(s):
[18145, 15649, 14303, 14493]
Open Access
Abstract: Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10–14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.
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May 2020
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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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Jonathan M.
Grimes
,
David R.
Hall
,
Alun W.
Ashton
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Gwyndaf
Evans
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Robin L.
Owen
,
Armin
Wagner
,
Katherine E.
Mcauley
,
Frank
Von Delft
,
Allen M.
Orville
,
Thomas
Sorensen
,
Martin A.
Walsh
,
Helen
Ginn
,
David I.
Stuart
Open Access
Abstract: Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide.
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Feb 2018
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Controls
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Abstract: Automation plays a key role in the macromolecular crystallography (MX) beamlines at Diamond Light Source (DLS). This is particularly evident with sample exchange; where fast, reliable, and accurate handling is required to ensure high quality and high throughput data collection. This paper looks at the design, build, and integration of an in-house robot control system. The system was designed to improve reliability and exchange times, provide high sample storage capacity, and accommodate easy upgrade paths, whilst gaining and maintaining in-house robotics knowledge. The paper also highlights how peripheral components were brought under the control of a Programmable Logic Controller (PLC) based integration unit, including a vision system.
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Jan 2018
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Data acquisition
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Abstract: In any experimental discipline, raw data represents the source from which all discoveries are derived. A more strict
interpretation in X-ray diffraction experiments may refer to this as primary data since any pixel counts will have been
manipulated (e.g. analogue to digital conversion, dark current correction, interpolation of pixels etc.); however the
fundamental idea remains: this is the closest it is possible to get to the original experimental measurements.
At Diamond Light Source, the principle of secure recording and storage of the primary data was embedded in the data
acquisition system from the outset. The general user does not have permission to alter or delete the raw experimental data,
and the acquisition system GDA is designed to prevent over-writing of the images.
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Aug 2017
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Abstract: SNi-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a β-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π–sugar interaction and reveal a quantum mechanics–molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, SNi-like glycosyltransferases. This observation of a front-face mechanism in a β-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'β-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.
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Jun 2017
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