I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18812]
Open Access
Abstract: Here, we have linked one of the most common protein-protein interaction events, homodimerisation, to an essential trace metal, copper, through engineering green fluorescent protein. Mutation of H148 to cysteine promotes the neutral phenolic chromophore in the monomer that excites predominantly at ~400 nm. Homodimerisation via a copper-dependent disulphide bridge, switches the chromophore to the charged phenolate that excites at ~490 nm. The result is ~30 fold increase in the fluorescence emission ratio. Homo-dimerisation kinetics are further improved by optimising the sfGFP homodimer interface, generating the variant termed GFP-diS2. Structures of the monomeric and dimeric GFP-diS2 suggests charge switching is through peptide bond flipping and the formation of a buried organised water network around the chromophore that span the interface region. Fusion to a leucine zipper protein dimerisation element greatly increased GFP-diS2 association rate making it a more effective copper sensor in vitro and in vivo with Cu(I) instigating the signal change quicker and at lower ion concentrations than Cu(II). Thus, GFP-diS2 provides the framework for generating a sensitive genetically encoded copper sensor and will eventually be adapted to monitor one of the most important protein-protein interactions in biology, homo-oligomerisation.
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Oct 2025
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B18-Core EXAFS
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Yinghao
Xu
,
Yi-Hsuan
Wu
,
Paula M.
Abdala
,
Connor
Sherwin
,
Veronica
Celorrio
,
Diana
Piankova
,
Payal
Chaudhary
,
Vitaly
Alexandrov
,
Agnieszka
Kierzkowska
,
Denis A.
Kuznetsov
,
Christoph R.
Müller
Diamond Proposal Number(s):
[36625]
Open Access
Abstract: Iridium-based oxides are among the most promising catalysts for the acidic oxygen evolution reaction (OER) owing to their high catalytic activity and stability. Substituting iridium with earth-abundant elements could lower costs and potentially boost its intrinsic activity even further; however, no unambiguous structure–activity relationships describing the physical origins of the effect of the substituent for this class of electrocatalysts have been established. In this work, we utilized a series of IrOx(:M) nanoparticle catalysts to correlate their in situ structural changes with intrinsic OER activity. We observe that IrOx(:M) with M = W and In feature a significantly higher Ir-mass-normalized OER activity than IrOx, however the activity enhancements have a different origin. While the increased activity of IrOx[thin space (1/6-em)]:[thin space (1/6-em)]In stems from a higher number of electrochemically active iridium centers (due to the leaching of indium), IrOx[thin space (1/6-em)]:[thin space (1/6-em)]W features a higher intrinsic OER activity compared to IrOx, due to electronic effects of W on neighboring Ir/O sites. Furthermore, operando electrochemical mass spectrometry experiments and density functional theory (DFT) calculations revealed that the enhanced OER activity of IrOx(:M) does not originate from a promotion of the lattice oxygen coupling mechanism, but is instead associated with a facilitated conventional adsorbate evolution mechanism.
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Oct 2025
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E02-JEM ARM 300CF
I09-Surface and Interface Structural Analysis
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Benedikt P.
Klein
,
Matthew A.
Stoodley
,
Joel
Deyerling
,
Luke A.
Rochford
,
Dylan B.
Morgan
,
David G.
Hopkinson
,
Sam
Sullivan-Allsop
,
Henry
Thake
,
Fulden
Eratam
,
Lars
Sattler
,
Sebastian M.
Weber
,
Gerhard
Hilt
,
Alexander
Generalov
,
Alexei
Preobrajenski
,
Thomas
Liddy
,
Leon B. S.
Williams
,
Mhairi A.
Buchan
,
Graham A
Rance
,
Tien-Lin
Lee
,
Alex
Saywell
,
Roman
Gorbachev
,
Sarah J.
Haigh
,
Christopher S.
Allen
,
Willi
Auwärter
,
Reinhard
Maurer
,
David A.
Duncan
Diamond Proposal Number(s):
[25379, 30875, 31695, 31165, 33709]
Open Access
Abstract: Chemical vapour deposition enables large-domain growth of ideal graphene, yet many applications of graphene require the controlled inclusion of specific defects. We present a one-step chemical vapour deposition procedure aimed at retaining the precursor topology when incorporated into the grown carbonaceous film. When azupyrene, the molecular analogue of the Stone–Wales defect in graphene, is used as a precursor, carbonaceous monolayers with a range of morphologies are produced as a function of the copper substrate growth temperature. The higher the substrate temperature during deposition, the closer the resulting monolayer is to ideal graphene. Analysis, with a set of complementary materials characterisation techniques, reveals morphological changes closely correlated with changes in the atomic adsorption heights, network topology, and concentration of 5-/7-membered carbon rings. The engineered defective carbon monolayers can be transferred to different substrates, potentially enabling applications in nanoelectronics, sensorics, and catalysis.
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Sep 2025
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B18-Core EXAFS
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Charlie A. F.
Nason
,
Ajay Piriya
Vijaya Kumar Saroja
,
Wanjun
Ren
,
Yingkangzi
Mei
,
Asma
Sarguroh
,
Yupei
Han
,
Yi
Lu
,
Jamie A.
Gould
,
Tim I.
Hyde
,
Veronica
Celorrio
,
Gopinathan
Sankar
,
Yang
Xu
Diamond Proposal Number(s):
[39009, 39790]
Open Access
Abstract: The ultimate goal of potassium-ion batteries (KIBs) is to become a serious competitor to lithium-ion batteries (LIBs). Achieving this requires the development of high energy density negative electrode materials, with transition metal oxides emerging as the most promising candidates. However, despite their high theoretical capacities, most transition metal oxides still struggle to achieve high performance, often necessitating substantial nanostructuring. Ion-exchange presents a facile and effective process for enhancing material properties, yet the demonstration of the exchanged ions undergoing redox activity has not been previously reported for KIBs. Herein, this work reports Ni0.25K0.5TiNbO5, synthesized through the ion-exchange between K+ and Ni2+, as a novel negative electrode material for KIBs. The ion-exchanged material achieves a specific capacity of 304 mAh g−1 in the first cycle and 162 mAh g−1 after 10 cycles, corresponding to a 240% and 156% increase compared to the pristine, unexchanged KTiNbO5 at the same cycle numbers. The structure–performance relationship was investigated in detail, shedding light on the previously unknown relationships between the level of hydration, degree of exchange and the performance of ion-exchanged materials. Furthermore, the exchanged Ni was demonstrated to be reversibly redox active, contributing to the observed capacity and representing a first for ion-exchanged materials in the KIB literature.
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Sep 2025
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I04-Macromolecular Crystallography
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Thomas D.
Downes
,
S. Paul
Jones
,
James D.
Firth
,
John F.
Darby
,
Amelia K.
Gilio
,
Hanna F.
Klein
,
Xinyu
Wang
,
David C
Blakemore
,
Claudia
De Fusco
,
Stephen D.
Roughley
,
Lewis R.
Vidler
,
Maria A.
Whatton
,
Alison Jo-Anne
Woolford
,
Gail L.
Wrigley
,
Roderick E.
Hubbard
,
Gideon
Davies
,
Peter
O'Brien
,
Liang
Wu
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Fragment-based drug discovery is widely used in both academia and industry during the early stages of drug discovery. There is a growing interest in the design of 3-D fragments for inclusion in fragment libraries in order to increase chemical space coverage. We present herein the design and synthesis of 58 shape-diverse 3-D fragments that are prepared using just three modular synthetic methodologies. The 3-D fragments comprise a cyclic scaffold (cyclopentane, pyrrolidine, piperidine, tetrahydrofuran or tetrahydropyran) with one aromatic or heteroaromatic ring and possess properties within 'rule-of-three' fragment space. 3-D shape is assessed using principal moments of inertia analysis and conformational diversity is achieved by considering all conformations up to 1.5 kcal mol -1 above the energy of the global minimum energy conformer. Due to the modular nature of the fragment syntheses, these 3-D fragments are synthetically-enabled for fragment elaboration followon work, a key design feature. This modular, shape-diverse 3-D fragment collection has delivered privileged starting points across a spectrum of targets. Fragments from the set have been crystallographically validated in the SARS-CoV-2 main protease (M pro ) and the nonstructural protein 3 (Nsp3) (Mac1) as well as human glycosyltransferase MGATV, a major enzyme in the mammalian N-glycosylation pathway and a promoter of aggressive metastatic cancers, underscoring the breadth of biological space that can be explored.
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Sep 2025
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I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Hans E.
Pfalzgraf
,
Aditya G.
Rao
,
Kakali
Sen
,
Hannah R.
Adams
,
Marcus
Edwards
,
You
Lu
,
Chin
Yong
,
Sofia
Jaho
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Sam
Horrell
,
James
Beilsten-Edmands
,
Robin L.
Owen
,
Colin R.
Andrew
,
Jonathan A. R.
Worrall
,
Ivo
Tews
,
Adrian J.
Mulholland
,
Michael A.
Hough
,
Thomas W.
Keal
Diamond Proposal Number(s):
[27313]
Open Access
Abstract: Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic c-type haem where the porphyrin is crosslinked to the protein via a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 haem oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460-containing hydroxylamine oxidoreductase utilises a tyrosine cross link to haem and performs similar chemistry. Previous crystal structures of cytochrome P460 from Nitrosomonas europaea (NeP460) clearly show the existence of a single crosslink between the NZ atom of lysine and the haem porphyrin, with mutagenesis studies indicating roles for the crosslink in positioning a proton transfer residue and/or influencing the distortion of the haem. Here we describe the evidence for a novel double crosslink between lysine and haem in the cytochrome P460 from Methylococcus capsulatus (Bath). In order to understand the complexities of this enzyme system we applied high resolution structural biology approaches at synchrotron and XFEL sources paired with crystal spectroscopies. Linked to this, we carried out QM/MM simulations that enabled the prediction of electronic absorption spectra providing a crucial validation to linking simulations and experimental structures. Our work demonstrates the feasibility of a double crosslink in McP460 and provides an opportunity to investigate how simulations can interact with experimental structures.
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Aug 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[37593, 31440]
Open Access
Abstract: Computational protein design is advancing rapidly. However, approaches and methods are needed to increase success rates and to elaborate designs. Here we describe the combination of rational and computational design to deliver three-helix bundle (3HB) peptide assemblies and single-chain proteins with control over topology and thermal stability. First, we garner sequence-to-structure relationships from antiparallel 3HBs in the Protein Data Bank. This gives core-packing rules, including layers of hydrogen-bonded polar residues, which are combined with surface-charge patterning to design complementary sequences for acidic (A), basic (B), and neutral (N) helices. By altering the design of the N helix, two sets of synthetic peptides are generated for clockwise and anticlockwise arrangements of the three-helix assemblies. Solution-phase characterisation shows that both ABN peptide mixtures form stable, heterotrimeric assemblies consistent with the targeted ‘up-down-up’ topologies. Next, AlphaFold2 models for both designs are used to seed computational designs of single-chain proteins where the helices are connected by loop building. Synthetic genes for these express in E. coli to yield soluble, monomeric, and thermally stable proteins. By systematically introducing additional polar layers within the core, the thermal stability of these proteins is varied without compromising the specificity of the helix-helix interactions. Chemical and thermal denaturation reveals comparable thermodynamic parameters to those of highly stable natural proteins. Four X-ray crystal structures confirm that the design models and AlphaFold2 predictions match to sub-Å accuracy.
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Aug 2025
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I24-Microfocus Macromolecular Crystallography
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S. Joe
Russell
,
Catherine R.
Back
,
Christopher
Perry
,
Kaiman A.
Cheung
,
Laurence
Maschio
,
Sacha N.
Charlton
,
Nicholas R.
Lees
,
Monserrat
Manzo-Ruiz
,
Martin A.
Hayes
,
Marc W.
Van Der Kamp
,
Paul R.
Race
,
Christine L.
Willis
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: The tetrodecamycins are tetracyclic natural products that exhibit potent antimicrobial activity against a multitude of drug-resistant pathogens. These compounds are structurally distinguished by the presence of a tetronate ring and trans-decalin with six contiguous asymmetric centres united by a seven-membered oxygen heterocycle. Herein we describe the first total synthesis of the antibiotic (−)-13-deoxytetrodecamycin. Our strategy is predicated on an enantioselective [4 + 2]-cycloaddition catalysed by the FAD-dependent Diels–Alderase TedJ, forming the trans-decalin with concomitant creation of two rings and four contiguous stereocenters with exquisite selectivity under mild conditions. In complementary studies, in vitro enzyme assays, X-ray crystallography and computational modelling are used to provide molecular insights into the TedJ catalysed reaction. These studies illustrate the power of adopting a chemoenzymatic approach for the enantioselective synthesis of a target compound which would be difficult to achieve using non-biological methods and provide a practical demonstration of the use of Diels–Alder biocatalysts in total synthesis. This approach has potentially widespread value in the global challenge of discovery and development of new antibiotics.
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Aug 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946, 19281]
Open Access
Abstract: Macrocyclization and multiple backbone N-methylations can significantly improve the pharmacological properties of peptides. Since chemical synthesis of such compounds is often challenging, enzyme-based production platforms are an interesting option. Here, we characterized OphP, a serine peptidase involved in the cyclization of omphalotins, a group of ribosomally produced dodecapeptides with multiple backbone N-methylations. OphP displays robust peptidase and macrocyclase activity towards multiply α-N-methylated peptides of various lengths and composition derived from the omphalotin precursor protein OphMA. In addition, OphP processes, with lower efficiency, peptides unrelated to OphMA, containing a MeGly, MeAla or Pro residue at the P1 site. Structural analysis reveals that OphP adopts a canonical prolyl oligopeptidase fold but, unlike other enzymes of this enzyme family, recognizes its substrates by their hydrophobic and multiply backbone N-methylated core rather than by the follower peptide. The activity of OphP could be harnessed for the enzymatic production of therapeutic peptides.
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Jul 2025
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I19-Small Molecule Single Crystal Diffraction
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Karen
Robertson
,
Mark R.
Warren
,
Lois E.
Wayment
,
Pollyanna
Payne
,
C. Daniel
Scott
,
Chick C.
Wilson
,
Lucy K.
Saunders
,
Graeme
Winter
,
Benjamin
Williams
,
Lauren E.
Hatcher
,
David R.
Allan
Diamond Proposal Number(s):
[22497, 21301]
Open Access
Abstract: Serial crystallography has revolutionalised our ability to analyse protein crystals; crystal structures can be uncovered by combining data from multiple crystals mitigating radiation damage through overexposure to the X-ray beam. With synchrotron sources becoming even brighter radiation damage is ever more pertinent for small molecule crystals as well. Combining serial crystallography with flow crystallisation, we pave the way to exploring high-throughput screening and kinetic studies, and application to small molecule crystals of up to mm-scale. Here we present the first known example of single crystal X-ray diffraction of small molecule crystals in a flow crystallisation environment. In situ single-crystal X-ray diffraction has been achieved on a series of growing singular crystals through use of segmented flow cooling crystallisation, holding crystals in the X-ray beam for 4.2 s whilst they freely rotate whilst flowing inside tubing. Upon triggering of a passing slug at the analysis point, the tubing is moved in the opposite direction to the flow, enabling the crystal to remain within the X-ray beam whilst maintaining the free rotation of the crystal due to fluid movement. Structure solution of paracetamol Form I has been achieved with 0.8 Å resolution using data combined from 13 crystals whilst unit cell information can be extracted from a single crystal.
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Jun 2025
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