I03-Macromolecular Crystallography
I23-Long wavelength MX
|
Céline
Zheng-Gérard
,
Jana
Joha
,
Maria
Carrasquero
,
Kamel
El Omari
,
Edward
Lowe
,
Shirish
Dubey
,
Simon J.
Draper
,
Yu-Chi
Chang
,
Hsi-Hsien
Lin
,
Alan D.
Salama
,
Kirsty
Mchugh
,
Elena
Seiradake
Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Granulomatosis with polyangiitis is a life-threatening systemic vasculitis, characterised by anti-neutrophil cytoplasmic autoantibodies (ANCA) most commonly against proteinase 3 (PR3), a protease expressed intracellularly and on the surface of neutrophils. Most cell surface PR3 is bound to the receptor CD177; however, the molecular mechanism of the interactions is not well understood. Here, we present crystal structures of CD177 in complex with PR3 and unliganded CD177. We describe a mainly hydrophobic binding interface between PR3 and CD177, involving the first two Ly6/uPAR (LU) domains of CD177. These form a globular structure which is connected to downstream domains via a flexible linker. Using a panel of PR3-ANCA-positive patient samples, we show that a significant proportion of ANCAs target the CD177-binding site of PR3 in these samples. Structure-guided mutation of the CD177-binding site on PR3 is effective in reducing PR3-ANCA binding. The results demonstrate that the CD177-binding surface of PR3 harbours a major PR3-ANCA epitope, and that the extent of binding to this surface varies between different patients.
|
Feb 2026
|
|
Aquilos-CryoFIB at Diamond
Krios III-Titan Krios III at Diamond
|
Diamond Proposal Number(s):
[29812]
Open Access
Abstract: Direct visualization of HIV-1 nuclear import through the nuclear pore complex (NPC) presents a technical challenge due to the rarity of this process. To enable systematic investigation, we developed a robust in situ system that mimics HIV-1 nuclear import in a near-native context using isolated HIV-1 virus like particles (VLP) cores and permeabilized CD4 + T lymphocyte (CEM) cells. This approach supports docking and translocation of abundant viral cores through nuclear pores into the nucleus. For high-resolution visualization, we implemented an integrated correlative approach to guide cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) imaging, enabling precise targeting and structural characterization of individual nuclear import events. Using this workflow, we visualized 510 HIV-1 VLP cores at distinct stages of nuclear import, capturing key snapshots of the full progression of nuclear import. Subsequent statistical and structural analyses allow classification of core morphologies and identification of translocation-associated remodeling in nuclear pores. This work provides a methodological foundation for dissecting HIV-1 and potentially other viruses nuclear import processes and post-entry events in a controlled and quantitative manner.
|
Aug 2025
|
|
B21-High Throughput SAXS
|
Open Access
Abstract: The Retriever complex recycles a wide range of transmembrane proteins from endosomes to the plasma membrane. The cargo adapter protein SNX17 has been implicated in recruiting the Retriever complex to endosomal membranes, yet the details of this interaction have remained elusive. Through biophysical and structural model-guided mutagenesis studies with recombinant proteins and liposomes, we have gained a deeper understanding of this process. Here, we demonstrate a direct interaction between SNX17 and Retriever, specifically between the C-terminal region of SNX17 and the interface of the Retriever subunits VPS35L and VPS26C. This interaction is enhanced upon the binding of SNX17 to its cargo in solution, due to the disruption of an intramolecular autoinhibitory interaction between the C-terminal region of SNX17 and the cargo binding pocket. In addition, SNX17 binding to membranes containing phosphatidylinositol-3-phosphate also promotes Retriever recruitment in a cargo-independent manner. Therefore, this work provides evidence of the dual activation mechanisms by which SNX17 modulates Retriever recruitment to the proximity of cargo and membranes, offering significant insights into the regulatory mechanisms of protein recycling at endosomes.
|
Dec 2024
|
|
Krios IV-Titan Krios IV at Diamond
|
Hannah
Wapenaar
,
Gillian
Clifford
,
Willow
Rolls
,
Moira
Pasquier
,
Hayden
Burdett
,
Yujie
Zhang
,
Gauri
Deak
,
Juan
Zou
,
Christos
Spanos
,
Mark R. D.
Taylor
,
Jacquie
Mills
,
James A.
Watson
,
Dhananjay
Kumar
,
Richard
Clark
,
Alakta
Das
,
Devisree
Valsakumar
,
Janice
Bramham
,
Philipp
Voigt
,
Duncan
Sproul
,
Marcus D.
Wilson
Diamond Proposal Number(s):
[24557, 31827]
Open Access
Abstract: DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns in vertebrates. Here we structurally and biochemically explore the interaction of DNMT3A1 with diverse modified nucleosomes indicative of different chromatin environments. A cryo-EM structure of the full-length DNMT3A1-DNMT3L complex with a H2AK119ub nucleosome reveals that the DNMT3A1 ubiquitin-dependent recruitment (UDR) motif interacts specifically with H2AK119ub and makes extensive contacts with the core nucleosome histone surface. This interaction facilitates robust DNMT3A1 binding to nucleosomes, and previously unexplained DNMT3A disease-associated mutations disrupt this interface. Furthermore, the UDR-nucleosome interaction synergises with other DNMT3A chromatin reading elements in the absence of histone ubiquitylation. H2AK119ub does not stimulate DNMT3A DNA methylation activity, as observed for the previously described H3K36me2 mark, which may explain low levels of DNA methylation on H2AK119ub marked facultative heterochromatin. This study highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome surface and increases our understanding of how DNMT3A1 chromatin recruitment occurs.
|
Nov 2024
|
|
Krios III-Titan Krios III at Diamond
|
Ana
Crespillo-Casado
,
Prathyush
Pothukuchi
,
Katerina
Naydenova
,
Matthew C. J.
Yip
,
Janet M.
Young
,
Jerome
Boulanger
,
Vimisha
Dharamdasani
,
Ceara
Harper
,
Pierre-Mehdi
Hammoudi
,
Elsje G.
Otten
,
Keith
Boyle
,
Mayuri
Gogoi
,
Harmit S.
Malik
,
Felix
Randow
Diamond Proposal Number(s):
[31336]
Open Access
Abstract: Innate immunity senses microbial ligands known as pathogen-associated molecular patterns (PAMPs). Except for nucleic acids, PAMPs are exceedingly taxa-specific, thus enabling pattern recognition receptors to detect cognate pathogens while ignoring others. How the E3 ubiquitin ligase RNF213 can respond to phylogenetically distant pathogens, including Gram-negative Salmonella, Gram-positive Listeria, and eukaryotic Toxoplasma, remains unknown. Here we report that the evolutionary history of RNF213 is indicative of repeated adaptation to diverse pathogen target structures, especially in and around its newly identified CBM20 carbohydrate-binding domain, which we have resolved by cryo-EM. We find that RNF213 forms coats on phylogenetically distant pathogens. ATP hydrolysis by RNF213’s dynein-like domain is essential for coat formation on all three pathogens studied as is RZ finger-mediated E3 ligase activity for bacteria. Coat formation is not diffusion-limited but instead relies on rate-limiting initiation events and subsequent cooperative incorporation of further RNF213 molecules. We conclude that RNF213 responds to evolutionarily distant pathogens through enzymatically amplified cooperative recruitment.
|
Oct 2024
|
|
B21-High Throughput SAXS
|
Reshma
Thamkachy
,
Bethan
Medina-Pritchard
,
Sang Ho
Park
,
Carla G.
Chiodi
,
Juan
Zou
,
Maria
De La Torre-Barranco
,
Kazuma
Shimanaka
,
Maria Alba
Abad
,
Cristina
Gallego Páramo
,
Regina
Feederle
,
Emilija
Ruksenaite
,
Patrick
Heun
,
Owen R.
Davies
,
Juri
Rappsilber
,
Dina
Schneidman-Duhovny
,
Uhn-Soo
Cho
,
A. Arockia
Jeyaprakash
Diamond Proposal Number(s):
[23510]
Open Access
Abstract: The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18β and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18β and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18β, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.
|
Jul 2024
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Jannik
Strauss
,
Craig
Wilkinson
,
Keni
Vidilaseris
,
Orquidea M.
De Castro Ribeiro
,
Jianing
Liu
,
James
Hillier
,
Maximilian
Wichert
,
Anssi M.
Malinen
,
Bernadette
Gehl
,
Lars J. C.
Jeuken
,
Arwen R.
Pearson
,
Adrian
Goldman
Open Access
Abstract: Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na+- and/or H+ across membranes to the hydrolysis of pyrophosphate. Their role in the virulence of protist pathogens like Plasmodium falciparum makes them an intriguing target for structural and functional studies. Here, we show the first structure of a K+-independent M-PPase, asymmetric and time-dependent substrate binding in time-resolved structures of a K+-dependent M-PPase and demonstrate pumping-before-hydrolysis by electrometric studies. We suggest how key residues in helix 12, 13, and the exit channel loops affect ion selectivity and K+-activation due to a complex interplay of residues that are involved in subunit-subunit communication. Our findings not only explain ion selectivity in M-PPases but also why they display half-of-the-sites reactivity. Based on this, we propose, for the first time, a unified model for ion-pumping, hydrolysis, and energy coupling in all M-PPases, including those that pump both Na+ and H+.
|
Jan 2024
|
|
I04-Macromolecular Crystallography
|
Benjamin F.
Cooper
,
Giedrė
Ratkevičiūtė
,
Luke A.
Clifton
,
Hannah
Johnston
,
Rachel
Holyfield
,
David J.
Hardy
,
Simon G.
Caulton
,
William
Chatterton
,
Pooja
Sridhar
,
Peter
Wotherspoon
,
Gareth W.
Hughes
,
Stephen C. L.
Hall
,
Andrew L.
Lovering
,
Timothy J.
Knowles
Diamond Proposal Number(s):
[26803]
Open Access
Abstract: The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.
|
Jan 2024
|
|
Krios IV-Titan Krios IV at Diamond
|
Diamond Proposal Number(s):
[26993]
Open Access
Abstract: Protein translocation across the endoplasmic reticulum (ER) membrane is an essential step during protein entry into the secretory pathway. The conserved Sec61 protein-conducting channel facilitates polypeptide translocation and coordinates cotranslational polypeptide-processing events. In cells, the majority of Sec61 is stably associated with a heterotetrameric membrane protein complex, the translocon-associated protein complex (TRAP), yet the mechanism by which TRAP assists in polypeptide translocation remains unknown. Here, we present the structure of the core Sec61/TRAP complex bound to a mammalian ribosome by cryogenic electron microscopy (cryo-EM). Ribosome interactions anchor the Sec61/TRAP complex in a conformation that renders the ER membrane locally thinner by significantly curving its lumenal leaflet. We propose that TRAP stabilizes the ribosome exit tunnel to assist nascent polypeptide insertion through Sec61 and provides a ratcheting mechanism into the ER lumen mediated by direct polypeptide interactions.
|
Nov 2023
|
|
|
|
Open Access
Abstract: Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage—late fusion—shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.
|
Mar 2023
|
|
