I04-Macromolecular Crystallography
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Takeshi
Nakama
,
Aaron
Wall
,
Garry
Dolton
,
Li-Rong
Tan
,
Hannah
Thomas
,
Hiroshi
Hamana
,
Yoshiki
Aritsu
,
Toong Seng
Tan
,
Mako
Toyoda
,
Yoshihiko
Goto
,
Huanyu
Li
,
Mizuki
Kitamatsu
,
Keiko
Udaka
,
Yusuke
Miyashita
,
Hiroyuki
Oshiumi
,
Kimitoshi
Nakamura
,
Yoji
Nagasaki
,
Rumi
Minami
,
Hirotomo
Nakata
,
Pierre J.
Rizkallah
,
Hiroyuki
Kishi
,
Takamasa
Ueno
,
Andrew K.
Sewell
,
Chihiro
Motozono
Open Access
Abstract: Cytotoxic T lymphocytes form a critical component of SARS-CoV-2 immunity by recognizing viral peptides bound to HLA class I molecules. Here, we identified the Spike-derived peptide NYNYLYRLF448-456 (NF9) as the immunodominant HLA-A*24:02-restricted epitope in both convalescent and vaccinated donors. Across cohorts, A24/NF9-specific responses were dominated by public TCR motifs featuring TRAV12-1 (or TRAV6-1) paired with TRBJ2-7 and a conserved CDR3β sequence (CASSXXXGYEQYF). Using a panel of thirteen TCRs, we mapped recognition of single amino acid substitutions within NF9 and identified residue 5 (L452) as the principal determinant of escape. The L452R substitution, characteristic of the Delta variant, abolished recognition across all tested TCRs despite preserved HLA binding. Crystallography of a representative public TCR (P1-15) revealed that mutation at position 5 reoriented the peptide within HLA-A*24:02, flipping the adjacent Y453 side chain into the peptide-binding groove and eliminating the dominant TCR contact. This position-5-driven conformational switch provided a structural mechanism for universal loss of NF9 recognition by HLA-A*24:02-restricted T-cells. Consistent with this, Delta-infected convalescents failed to mount de novo NF9-5R-specific responses while retaining responses to the conserved A24/QI9 Spike epitope. Together, these findings defined the basis of A24/NF9 recognition and showed how one mutation remodelled peptide presentation to abrogate TCR responses.
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Mar 2026
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I23-Long wavelength MX
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Hannah
Best
,
Lainey J.
Williamson
,
Adam B.
Cutts
,
Marina
Galchenkova
,
Oleksandr
Yefanov
,
Nicole
Bryce-Sharron
,
Emily A.
Heath
,
Raphael
De Wijn
,
Robin
Schubert
,
Anna
Munke
,
Alessandra
Henkel
,
Bjarne
Klopprogge
,
T. Emilie S.
Scheer
,
Viviane
Kremling
,
Salah
Awel
,
Gisel
Pena
,
Juraj
Knoska
,
Anusha
Keloth
,
Julia
Maracke
,
Romain
Letrun
,
Egor
Sobolev
,
Johan
Bielecki
,
Diogo
Melo
,
Sravya
Kantamneni
,
Katerina
Doerner
,
Marco
Kloos
,
Joachim
Schulz
,
P. Lourdu
Xavier
,
Marius
Lauffer
,
Maite
Villanueva
,
Primitivo
Caballero
,
Helen
Waller-Evans
,
Emyr
Lloyd-Evans
,
Charlotte
Uetrecht
,
Richard
Bean
,
Henry N.
Chapman
,
Neil
Crickmore
,
Pierre J.
Rizkallah
,
Colin
Berry
,
Dominik
Oberthuer
Diamond Proposal Number(s):
[36446]
Open Access
Abstract: Bacillus thuringiensis (Bt) strains naturally produce pesticidal proteins as nanocrystalline inclusions that are extraordinarily stable in aqueous environments, but which dissolve selectively at specific pH conditions. These proteins have been used in agriculture for >50 years and are critical to global food security. The majority of previously determined Bt Cry protein structures lack the extended C-terminal “crystallization domain,” which is thought to stabilize crystal packing and control selective solubility in insect targets, often via manipulation of disulfide bridges. It has also recently been shown to influence toxicity and target specificity. Here, we use serial femtosecond crystallography (SFX) to determine high-resolution full-length native structures of Cry1Ca18 (1.65 Å) and Cry8Ba2 (2.27 Å) in their natural nanocrystalline state. Differences in cysteine content (19 versus 4 residues) reveal distinct in vivo crystal-stabilization strategies. Understanding Bt toxin domain architecture and natural crystal formation is essential for improving biopesticide design and advancing agricultural genetic engineering.
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Feb 2026
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I03-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Diamond Proposal Number(s):
[29990, 36446]
Open Access
Abstract: The red fluorescent protein mCherry is one of the most widely used fluorescent proteins in biology. Here, we have changed the chromophore chemistry by converting the thioether group of M66 to a thiol group through mutation to cysteine. The new variant, termed mCoral (due to its orange fluorescence hue), has similar brightness to mCherry but improved resistance to hydrogen peroxide. The variant is also responsive to pH with low and high pKa forms that have distinct spectral properties, which DFT analysis suggests is due to protonation state changes in the newly introduced thiol group, as well as the phenol group. The structure of mCoral reveals that the M66C mutation creates a space within the β-barrel structure that is filled by a water molecule, which makes new polar interactions, including the backbone carbonyl group of F65. Molecular dynamics simulations suggest that this additional water molecule, together with local solvation around the chromophore, could play a role in promoting planarity of the full conjugated system comprising the chromophore. The mCoral chromophore makes slightly more H-bonds with water than mCherry. The main water exit point for mCherry is also narrower in mCoral, providing a potential explanation for increased resistance to hydrogen peroxide. Overall, a small structural change to mCherry has resulted in a new fluorescent protein with potentially useful characteristics and an insight into the role of dynamics and water in defining the structure–function relationship in red fluorescent proteins.
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Dec 2025
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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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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Garry
Dolton
,
Anna
Bulek
,
Aaron
Wall
,
Hannah
Thomas
,
Jade R.
Hopkins
,
Cristina
Rius
,
Sarah A. E.
Galloway
,
Thomas
Whalley
,
Li Rong
Tan
,
Théo
Morin
,
Nader
Omidvar
,
Anna
Fuller
,
Katie
Topley
,
Md Samiul
Hasan
,
Shikha
Jain
,
Nirupa
D’souza
,
Thomas
Hodges-Hoyland
,
Owen B.
Spiller
,
Deborah
Kronenberg-Versteeg
,
Barbara
Szomolay
,
Hugo A.
Van Den Berg
,
Lucy C.
Jones
,
Mark
Peakman
,
David K.
Cole
,
Pierre J.
Rizkallah
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[10462, 18812]
Open Access
Abstract: CD8+ T cells destroy insulin-producing pancreatic β cells in type 1 diabetes through HLA class I–restricted presentation of self-antigens. Combinatorial peptide library screening was used to produce a preferred peptide recognition landscape for a patient-derived T cell receptor (TCR) that recognized the preproinsulin-derived (PPI-derived) peptide sequence LWMRLLPLL in the context of disease risk allele HLA A*24:02. Data were used to generate a strong superagonist peptide, enabling production of an autoimmune HLA A*24:02–peptide–TCR structure by crystal seeding. TCR binding to the PPI epitope was strongly focused on peptide residues Arg4 and Leu5, with more flexibility at other positions, allowing the TCR to strongly engage many peptides derived from pathogenic bacteria. We confirmed an epitope from Klebsiella that was recognized by PPI-reactive T cells from 3 of 3 HLA A*24:02+ patients. Remarkably, the same epitope selected T cells from 7 of 8 HLA A*24+ healthy donors that cross-reacted with PPI, leading to recognition and killing of HLA A*24:02+ cells expressing PPI. These data provide a mechanism by which molecular mimicry between pathogen and self-antigens could have resulted in the breaking of self-tolerance to initiate disease.
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Sep 2024
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I03-Macromolecular Crystallography
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Sarah
Hulin-Curtis
,
James K.
Geary
,
Bruce J.
Maclachlan
,
Danny M.
Altmann
,
Laury
Baillon
,
David K.
Cole
,
Alexander
Greenshields-Watson
,
Sophie J.
Hesketh
,
Ian R.
Humphreys
,
Ian M.
Jones
,
Sarah N.
Lauder
,
Georgina H.
Mason
,
Kathryn
Smart
,
D. Oliver
Scourfield
,
Jake
Scott
,
Ksenia
Sukhova
,
Richard J.
Stanton
,
Aaron
Wall
,
Pierre J.
Rizkallah
,
Wendy S.
Barclay
,
Awen
Gallimore
,
Andrew
Godkin
Diamond Proposal Number(s):
[10462]
Open Access
Abstract: CD4+ T cells are central to adaptive immunity. Their role in cross-protection in viral infections such as influenza and severe acute respiratory syndrome (SARS) is well documented; however, molecular rules governing T cell receptor (TCR) engagement of peptide-human leukocyte antigen (pHLA) class II are less understood. Here, we exploit an aspect of HLA class II presentation, the peptide-flanking residues (PFRs), to “tune” CD4+ T cell responses within an in vivo model system of influenza. Using a recombinant virus containing targeted substitutions at immunodominant HLA-DR1 epitopes, we demonstrate limited weight loss and improved clinical scores after heterosubtypic re-challenge. We observe enhanced protection linked to lung-derived influenza-specific CD4+ and CD8+ T cells prior to re-infection. Structural analysis of the ternary TCR:pHLA complex identifies that flanking amino acids influence side chains in the core 9-mer peptide, increasing TCR affinity. Augmentation of CD4+ T cell immunity is achievable with a single mutation, representing a strategy to enhance adaptive immunity that is decoupled from vaccine modality.
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Jun 2024
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18812, 20147, 29990]
Open Access
Abstract: Human adenoviruses (HAdV) are widespread pathogens causing usually mild infections. The Species D (HAdV-D) cause gastrointestinal tract infections and epidemic keratoconjunctivitis (EKC). Despite being significant pathogens, knowledge around HAdV-D mechanism of cell infection is lacking. Sialic acid (SA) usage has been proposed as a cell infection mechanism for EKC causing HAdV-D. Here we highlight an important role for SA engagement by many HAdV-D. We provide apo state crystal structures of 7 previously undetermined HAdV-D fiber-knob proteins, and structures of HAdV-D25, D29, D30 and D53 fiber-knob proteins in complex with SA. Biologically, we demonstrate that removal of cell surface SA reduced infectivity of HAdV-C5 vectors pseudotyped with HAdV-D fiber-knob proteins, whilst engagement of the classical HAdV receptor CAR was variable. Our data indicates variable usage of SA and CAR across HAdV-D. Better defining these interactions will enable improved development of antivirals and engineering of the viruses into refined therapeutic vectors.
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Sep 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yuan
Chen
,
Georgina H.
Mason
,
D. Oliver
Scourfield
,
Alexander
Greenshields-Watson
,
Tracey A.
Haigh
,
Andrew K.
Sewell
,
Heather M.
Long
,
Awen M.
Gallimore
,
Pierre
Rizkallah
,
Bruce J.
Maclachlan
,
Andrew
Godkin
Diamond Proposal Number(s):
[20147, 29502, 29990]
Open Access
Abstract: CD4+ T cells recognize a broad range of peptide epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which contribute to immune memory and limit COVID-19 disease. We demonstrate that the immunogenicity of SARS-CoV-2 peptides, in the context of the model allotype HLA-DR1, does not correlate with their binding affinity to the HLA heterodimer. Analyzing six epitopes, some with very low binding affinity, we solve X-ray crystallographic structures of each bound to HLA-DR1. Further structural definitions reveal the precise molecular impact of viral variant mutations on epitope presentation. Omicron escaped ancestral SARS-CoV-2 immunity to two epitopes through two distinct mechanisms: (1) mutations to TCR-facing epitope positions and (2) a mechanism whereby a single amino acid substitution caused a register shift within the HLA binding groove, completely altering the peptide-HLA structure. This HLA-II-specific paradigm of immune escape highlights how CD4+ T cell memory is finely poised at the level of peptide-HLA-II presentation.
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Aug 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Garry
Dolton
,
Cristina
Rius
,
Aaron
Wall
,
Barbara
Szomolay
,
Valentina
Bianchi
,
Sarah A. E.
Galloway
,
Md Samiul
Hasan
,
Théo
Morin
,
Marine E.
Caillaud
,
Hannah L.
Thomas
,
Sarah
Theaker
,
Li Rong
Tan
,
Anna
Fuller
,
Katie
Topley
,
Mateusz
Legut
,
Meriem
Attaf
,
Jade R.
Hopkins
,
Enas
Behiry
,
Joanna
Zabkiewicz
,
Caroline
Alvares
,
Angharad
Lloyd
,
Amber
Rogers
,
Peter
Henley
,
Christopher
Fegan
,
Oliver
Ottmann
,
Stephen
Man
,
Michael D.
Crowther
,
Marco
Donia
,
Inge Marie
Svane
,
David K.
Cole
,
Paul E.
Brown
,
Pierre
Rizkallah
,
Andrew K.
Sewell
Open Access
Abstract: The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such “multipronged” T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.
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Jul 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[20147]
Open Access
Abstract: Tpp80Aa1 from Bacillus thuringiensis is a Toxin_10 family protein (Tpp) with reported action against Culex mosquitoes. Here, we demonstrate an expanded target range, showing Tpp80Aa1 is also active against the larvae of Anopheles gambiae and Aedes aegypti mosquitoes. We report the first crystal structure of Tpp80Aa1 at a resolution of 1.8 Å, which shows Tpp80Aa1 consists of two domains: an N-terminal β-trefoil domain resembling a ricin B lectin and a C-terminal putative pore-forming domain sharing structural similarity with the aerolysin family. Similar to other Tpp family members, we observe Tpp80Aa1 binds to the mosquito midgut, specifically the posterior midgut and the gastric caecum. We also identify that Tpp80Aa1 can interact with galactose-containing glycolipids and galactose, and this interaction is critical for exerting full insecticidal action against mosquito target cell lines.
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Dec 2022
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