I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[1087, 8889]
Open Access
Abstract: Capsular polysaccharides (CPSs) are protective structures on the surfaces of many Gram-negative bacteria. The principal CPS of the human pathogen and Tier 1 select agent Burkholderia pseudomallei consists of a linear repeat of –3)-2-O-acetyl-6-deoxy-β-D-manno-heptopyranose-(1–. This CPS is critical to the virulence of this emerging pathogen and represents a key target for the development of novel therapeutics. wcbI is one of several genes in the CPS biosynthetic cluster whose deletion leads to significant attenuation of the pathogen; unlike most others, it has no homologues of known function and no detectable sequence similarity to any protein with an extant structure. Here, the crystal structure of WcbI bound to its proposed product, coenzyme A, is reported at 1.38 Å resolution, solved using the halide-soak method with multiple anomalous dispersion. This structure reveals that WcbI incorporates a previously described 100-amino-acid subdomain into a novel, principally helical fold (310 amino acids). This fold adopts a cradle-like structure, with a deep binding pocket for CoA in the loop-rich cradle. Structural analysis and biophysical assays suggest that WcbI functions as an acetyltransferase enzyme, whilst biochemical tests suggest that another functional module might be required to assist its activity in forming the mature B. pseudomallei capsule.
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Oct 2013
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8302]
Open Access
Abstract: S-Adenosylmethionine (SAMe) is the principal methyl donor of the cell and is
synthesized via an ATP-driven process by methionine adenosyltransferase
(MAT) enzymes. It is tightly linked with cell proliferation in liver and colon
cancer. In humans, there are three genes, mat1A, mat2A and mat2B, which
encode MATenzymes. mat2A and mat2B transcribe MATa2 and MATb enzyme
subunits, respectively, with catalytic and regulatory roles. The MATa2b complex
is expressed in nearly all tissues and is thought to be essential in providing the
necessary SAMe flux for methylation of DNA and various proteins including
histones. In human hepatocellular carcinoma mat2A and mat2B genes are
upregulated, highlighting the importance of the MATa2b complex in liver
disease. The individual subunits have been structurally characterized but the
nature of the complex has remained elusive despite its existence having been
postulated for more than 20 years and the observation that MATb is often colocalized
with MATa2. Though SAMe can be produced by MAT(a2)4 alone, this
paper shows that the Vmax of the MATa2b complex is three- to fourfold higher
depending on the variants of MATb that participate in complex formation.
Using X-ray crystallography and solution X-ray scattering, the first structures
are provided of this 258 kDa functional complex both in crystals and solution
with an unexpected stoichiometry of 4a2 and 2bV2 subunits. It is demonstrated
that the N-terminal regulates the activity of the complex and it is shown that
complex formation takes place surprisingly via the C-terminal of MATbV2 that
buries itself in a tunnel created at the interface of the MAT(a2)2. The structural
data suggest a unique mechanism of regulation and provide a gateway for
structure-based drug design in anticancer therapies.
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Jul 2014
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7864]
Open Access
Abstract: The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed. N-Myristoyltransferase (NMT) has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT from Leishmania donovani have been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely related L. major are reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic ?-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed.
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Jul 2014
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Open Access
Abstract: Small- and wide-angle X-ray scattering (SAXS, WAXS) are standard tools in materials research. The simultaneous measurement of SAXS and WAXS data in time-resolved studies has gained popularity due to the complementary information obtained. Furthermore, the combination of these data with non X-ray based techniques, via either simultaneous or independent measurements, has advanced understanding of the driving forces that lead to the structures and morphologies of materials, which in turn give rise to their properties. The simultaneous measurement of different data regimes and types, using either X-rays or neutrons, and the desire to control parameters that initiate and control structural changes have led to greater demands on sample environments. Examples of developments in technique combinations and sample environment design are discussed, together with a brief speculation about promising future developments
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Nov 2014
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Open Access
Abstract: Human transthyretin has an intrinsic tendency to form amyloid fibrils and is heavily implicated in senile systemic amyloidosis. Here, detailed neutron structural studies of perdeuterated transthyretin are described. The analyses, which fully exploit the enhanced visibility of isotopically replaced hydrogen atoms, yield new information on the stability of the protein and the possible mechanisms of amyloid formation. Residue Ser117 may play a pivotal role in that a single water molecule is closely associated with the γ-hydrogen atoms in one of the binding pockets, and could be important in determining which of the two sites is available to the substrate. The hydrogen-bond network at the monomer–monomer interface is more extensive than that at the dimer–dimer interface. Additionally, the edge strands of the primary dimer are seen to be favourable for continuation of the β-sheet and the formation of an extended cross-β structure through sequential dimer couplings. It is argued that the precursor to fibril formation is the dimeric form of the protein.
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Nov 2014
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I11-High Resolution Powder Diffraction
I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[9972]
Open Access
Abstract: A family of one-dimensional coordination polymers, [Ag4(O2C(CF2)2CF3)4(phenazine)2(arene)n]·m(arene), 1 (arene = toluene or xylene), have been synthesized and crystallographically characterized. Arene guest loss invokes structural transformations to yield a pair of polymorphic coordination polymers [Ag4(O2C(CF2)2CF3)4(phenazine)2], 2a and/or 2b, with one- and two-dimensional architectures, respectively. The role of pre-organization of the polymer chains of 1 in the selectivity for formation of either polymorph is explored, and the templating effect of toluene and p-xylene over o-xylene or m-xylene in the formation of arene-containing architecture 1 is also demonstrated. The formation of arene-free phase 2b, not accessible in a phase-pure form through other means, is shown to be the sole product of loss of toluene from 1-tol·tol [Ag4(O2C(CF2)2CF3)4(phenazine)2(toluene)]·2(toluene), a phase containing toluene coordinated to Ag(I) in an unusual ?:?1,?1 manner. Solvent-vapour-assisted conversion between the polymorphic coordination polymers and solvent-vapour influence on the conversion of coordination polymers 1 to 2a and 2b is also explored. The transformations have been examined and confirmed by X-ray diffraction, NMR spectroscopy and thermal analyses, including in situ diffraction studies of some transformations.
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Mar 2015
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NONE-No attached Diamond beamline
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Przemyslaw
Nogly
,
Daniel
James
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Dingjie
Wang
,
Thomas A.
White
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Nadia
Zatsepin
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Anastasya
Shilova
,
Garrett
Nelson
,
Haiguang
Liu
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Linda
Johansson
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Michael
Heymann
,
Kathrin
Jaeger
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Markus
Metz
,
Cecilia
Wickstrand
,
Wenting
Wu
,
Petra
Båth
,
Peter
Berntsen
,
Dominik
Oberthuer
,
Valerie
Panneels
,
Vadim
Cherezov
,
Isabel
Moraes
,
Henry
Chapman
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Gebhard
Schertler
,
Richard
Neutze
,
John
Spence
,
Manfred
Burghammer
,
Joerg
Standfuss
,
Uwe
Weierstall
Open Access
Abstract: Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.
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Mar 2015
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Data acquisition
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Open Access
Abstract: Native SAD phasing uses the anomalous scattering signal of light atoms in the crystalline, native samples of macromolecules collected from single-wavelength X-ray diffraction experiments. These atoms include sodium, magnesium, phosphorus, sulfur, chlorine, potassium and calcium. Native SAD phasing is challenging and is critically dependent on the collection of accurate data. Over the past five years, advances in diffraction hardware, crystallographic software, data-collection methods and strategies, and the use of data statistics have been witnessed which allow `highly accurate data' to be routinely collected. Today, native SAD sits on the verge of becoming a `first-choice' method for both de novo and molecular-replacement structure determination. This article will focus on advances that have caught the attention of the community over the past five years. It will also highlight both de novo native SAD structures and recent structures that were key to methods development.
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Jul 2015
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11740]
Open Access
Abstract: Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07–1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a `catalytic reaction movie' highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines.
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Jul 2016
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I19-Small Molecule Single Crystal Diffraction
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Christopher H.
Woodall
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Jeppe
Christensen
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Jonathan M.
Skelton
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Lauren E.
Hatcher
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Andrew
Parlett
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Paul R.
Raithby
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Aron
Walsh
,
Stephen C.
Parker
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Christine M.
Beavers
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Simon J.
Teat
,
Mourad
Intissar
,
Christian
Reber
,
David R.
Allan
Open Access
Abstract: We report a molecular crystal that exhibits four successive phase transitions under hydrostatic pressure, driven by aurophilic interactions, with the ground-state structure re-emerging at high pressure. The effect of pressure on two polytypes of tris(μ2-3,5-diisopropyl-1,2,4-triazolato-κ2N1:N2)trigold(I) (denoted Form-I and Form-II) has been analysed using luminescence spectroscopy, single-crystal X-ray diffraction and first-principles computation. A unique phase behaviour was observed in Form-I, with a complex sequence of phase transitions between 1 and 3.5 GPa. The ambient C2/c mother cell transforms to a P21/n phase above 1 GPa, followed by a P21/a phase above 2 GPa and a large-volume C2/c supercell at 2.70 GPa, with the previously observed P21/n phase then reappearing at higher pressure. The observation of crystallographically identical low- and high-pressure P21/n phases makes this a rare example of a re-entrant phase transformation. The phase behaviour has been characterized using detailed crystallographic theory and modelling, and rationalized in terms of molecular structural distortions. The dramatic changes in conformation are correlated with shifts of the luminescence maxima, from a band maximum at 14040 cm−1 at 2.40 GPa, decreasing steeply to 13550 cm−1 at 3 GPa. A similar study of Form-II displays more conventional crystallographic behaviour, indicating that the complex behaviour observed in Form-I is likely to be a direct consequence of the differences in crystal packing between the two polytypes.
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Sep 2016
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