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Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Simultaneous binding of guidance cues NET1 and RGM blocks extracellular NEO1 signaling Ross A. Robinson , Samuel C. Griffiths , Lieke L. Van De Haar , Tomas Malinauskas , Eljo Y. Van Battum , Pavol Zelina , Rebekka A. Schwab , Dimple Karia , Lina Malinauskaite , Sara Brignani , Marleen H. Van Den Munkhof , Özge Düdükcü , Anna A. De Ruiter , Dianne M.a. Van Den Heuvel , Benjamin Bishop , Jonathan Elegheert , A. Radu Aricescu , R. Jeroen Pasterkamp , Christian Siebold Cell 74 DOI: 10.1016/j.cell.2021.02.045 Open Access Show Abstract ▼ Abstract: During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a “trimer-of-trimers” super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.	Mar 2021
	A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses Tiong Kit Tan , Pramila Rijal , Rolle Rahikainen , Anthony H. Keeble , Lisa Schimanski , Saira Hussain , Ruth Harvey , Jack W. P. Hayes , Jane C. Edwards , Rebecca K. Mclean , Veronica Martini , Miriam Pedrera , Nazia Thakur , Carina Conceicao , Isabelle Dietrich , Holly Shelton , Anna Ludi , Ginette Wilsden , Clare Browning , Adrian K. Zagrajek , Dagmara Bialy , Sushant Bhat , Phoebe Stevenson-leggett , Philippa Hollinghurst , Matthew Tully , Katy Moffat , Chris Chiu , Ryan Waters , Ashley Gray , Mehreen Azhar , Valerie Mioulet , Joseph Newman , Amin S. Asfor , Alison Burman , Sylvia Crossley , John A. Hammond , Elma Tchilian , Bryan Charleston , Dalan Bailey , Tobias J. Tuthill , Simon P. Graham , Helen M. E. Duyvesteyn , Tomas Malinauskas , Jiandong Huo , Julia A. Tree , Karen R. Buttigieg , Raymond J. Owens , Miles W. Carroll , Rodney S. Daniels , John W. Mccauley , David I. Stuart , Kuan-ying A. Huang , Mark Howarth , Alain R. Townsend Nature Communications 12 DOI: 10.1038/s41467-020-20654-7 Open Access Show Abstract ▼ Abstract: There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.	Jan 2021
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2 Jiangdong Huo , Audrey Le Bas , Reinis R. Ruza , Helen M. E. Duyvesteyn , Halina Mikolajek , Tomas Malinauskas , Tiong Kit Tan , Pramila Rijal , Maud Dumoux , Philip N. Ward , Jingshan Ren , Daming Zhou , Peter J. Harrison , Miriam Weckener , Daniel K. Clare , Vinod K. Vogirala , Julika Radecke , Lucile Moynie , Yuguang Zhao , Javier Gilbert-jaramillo , Michael L. Knight , Julia A. Tree , Karen R. Buttigieg , Naomi Coombes , Michael J. Elmore , Miles W. Carroll , Loic Carrique , Pranav N. M. Shah , William James , Alain R. Townsend , David I. Stuart , Raymond J. Owens , James H. Naismith Nature Structural & Molecular Biology 21 DOI: 10.1038/s41594-020-0469-6 Diamond Proposal Number(s): [27031, 27051] Open Access Show Abstract ▼ Abstract: The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody–RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD–ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4–6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.	Jul 2020
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Glypicans shield the Wnt lipid moiety to enable signalling at a distance Ian J. Mcgough , Luca Vecchia , Benjamin Bishop , Tomas Malinauskas , Karen Beckett , Dhira Joshi , Nicola O’reilly , Christian Siebold , E. Yvonne Jones , Jean-paul Vincent Nature 423 DOI: 10.1038/s41586-020-2498-z Diamond Proposal Number(s): [19946] Show Abstract ▼ Abstract: A relatively small number of proteins have been suggested to act as morphogens—signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1,2,3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4,5,6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.	Jul 2020
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient Daming Zhou , Helen M. E. Duyvesteyn , Cheng-pin Chen , Chung-guei Huang , Ting-hua Chen , Shin-ru Shih , Yi-chun Lin , Chien-yu Cheng , Shu-hsing Cheng , Yhu-chering Huang , Tzou-yien Lin , Che Ma , Jiandong Huo , Loic Carrique , Tomas Malinauskas , Reinis R. Ruza , Pranav Shah , Tiong Kit Tan , Pramila Rijal , Robert F. Donat , Kerry Godwin , Karen R. Buttigieg , Julia A. Tree , Julika Radecke , Neil Paterson , Piyada Supasa , Juthathip Mongkolsapaya , Gavin R. Screaton , Miles W. Carroll , Javier Gilbert-jaramillo , Michael L. Knight , William James , Raymond J. Owens , James H. Naismith , Alain R. Townsend , Elizabeth E. Fry , Yuguang Zhao , Jingshan Ren , David I. Stuart , Kuan-ying A. Huang Nature Structural & Molecular Biology 20 DOI: 10.1038/s41594-020-0480-y Diamond Proposal Number(s): [19946, 26983] Show Abstract ▼ Abstract: The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD–EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.	Jul 2020
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Neutralisation of SARS-CoV-2 by destruction of the prefusion Spike Jiandong Huo , Yuguang Zhao , Jingshan Ren , Daming Zhou , Helen M. E. Duyvesteyn , Helen M. Ginn , Loic Carrique , Tomas Malinauskas , Reinis R. Ruza , Pranav N. M. Shah , Tiong Kit Tan , Pramila Rijal , Naomi Coombes , Kevin R. Bewley , Julia A. Tree , Julika Radecke , Neil Paterson , Piyasa Supasa , Juthathip Mongkolsapaya , Gavin R. Screaton , Miles Carroll , Alain Townsend , Elizabeth E. Fry , Raymond J. Owens , David I. Stuart Cell Host & Microbe DOI: 10.1016/j.chom.2020.06.010 Diamond Proposal Number(s): [19946, 26983] Open Access Show Abstract ▼ Abstract: There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognises angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment.	Jun 2020
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Repulsive guidance molecules lock growth differentiation factor 5 in an inhibitory complex Tomas Malinauskas , Tina V. Peer , Benjamin Bishop , Thomas D. Mueller , Christian Siebold Proceedings Of The National Academy Of Sciences 22 DOI: 10.1073/pnas.2000561117 Diamond Proposal Number(s): [14744, 10627] Open Access Show Abstract ▼ Abstract: Repulsive guidance molecules (RGMs) are cell surface proteins that regulate the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. They control fundamental biological processes, such as migration and differentiation by direct interaction with the Neogenin (NEO1) receptor and function as coreceptors for the bone morphogenetic protein (BMP)/growth differentiation factor (GDF) family. We determined crystal structures of all three human RGM family members in complex with GDF5, as well as the ternary NEO1–RGMB–GDF5 assembly. Surprisingly, we show that all three RGMs inhibit GDF5 signaling, which is in stark contrast to RGM-mediated enhancement of signaling observed for other BMPs, like BMP2. Despite their opposite effect on GDF5 signaling, RGMs occupy the BMP type 1 receptor binding site similar to the observed interactions in RGM–BMP2 complexes. In the NEO1–RGMB–GDF5 complex, RGMB physically bridges NEO1 and GDF5, suggesting cross-talk between the GDF5 and NEO1 signaling pathways. Our crystal structures, combined with structure-guided mutagenesis of RGMs and BMP ligands, binding studies, and cellular assays suggest that RGMs inhibit GDF5 signaling by competing with GDF5 type 1 receptors. While our crystal structure analysis and in vitro binding data initially pointed towards a simple competition mechanism between RGMs and type 1 receptors as a possible basis for RGM-mediated GDF5 inhibition, further experiments utilizing BMP2-mimicking GDF5 variants clearly indicate a more complex mechanism that explains how RGMs can act as a functionality-changing switch for two structurally and biochemically similar signaling molecules.	Jun 2020
I23-Long wavelength MX	The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism Amalie F. Rudolf , Maia Kinnebrew , Christiane Kowatsch , T. Bertie Ansell , Kamel El Omari , Benjamin Bishop , Els Pardon , Rebekka A. Schwab , Tomas Malinauskas , Mingxing Qian , Ramona Duman , Douglas F. Covey , Jan Steyaert , Armin Wagner , Mark S. P. Sansom , Rajat Rohatgi , Christian Siebold Nature Chemical Biology 15, 975 - 982 DOI: 10.1038/s41589-019-0370-y Show Abstract ▼ Abstract: Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids—a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.	Oct 2019
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Diversity of oligomerization in Drosophila semaphorins suggests a mechanism of functional fine-tuning Daniel Rozbesky , Ross A. Robinson , Vitul Jain , Max Renner , Tomas Malinauskas , Karl Harlos , Christian Siebold , E. Yvonne Jones Nature Communications 10 DOI: 10.1038/s41467-019-11683-y Diamond Proposal Number(s): [14744, 10627] Open Access Show Abstract ▼ Abstract: Semaphorin ligands and their plexin receptors are one of the major cell guidance factors that trigger localised changes in the cytoskeleton. Binding of semaphorin homodimer to plexin brings two plexins in close proximity which is a prerequisite for plexin signalling. This model appears to be too simplistic to explain the complexity and functional versatility of these molecules. Here, we determine crystal structures for all members of Drosophila class 1 and 2 semaphorins. Unlike previously reported semaphorin structures, Sema1a, Sema2a and Sema2b show stabilisation of sema domain dimer formation via a disulfide bond. Unexpectedly, our structural and biophysical data show Sema1b is a monomer suggesting that semaphorin function may not be restricted to dimers. We demonstrate that semaphorins can form heterodimers with members of the same semaphorin class. This heterodimerization provides a potential mechanism for cross-talk between different plexins and co-receptors to allow fine-tuning of cell signalling.	Aug 2019
Krios I-Titan Krios I at Diamond	GABAA receptor signalling mechanisms revealed by structural pharmacology Simonas Masiulis , Rooma Desai , Tomasz Uchanski , Itziar Serna Martin , Duncan Laverty , Dimple Karia , Tomas Malinauskas , Jasenko Zivanov , Els Pardon , Abhay Kotecha , Jan Steyaert , Keith W. Miller , A. Radu Aricescu Nature 565, 454 - 459 DOI: 10.1038/s41586-018-0832-5 Show Abstract ▼ Abstract: Type-A γ-aminobutyric (GABAA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABAA receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABAA receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABAA receptor modulators.	Jan 2019

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