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Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond Krios IV-Titan Krios IV at Diamond	Neutral sphingomyelinase 2 is required for HIV-1 maturation Abdul A. Waheed , Yanan Zhu , Eva Agostino , Lwar Naing , Yuta Hikichi , Ferri Soheilian , Seung-Wan Yoo , Yun Song , Peijun Zhang , Barbara S. Slusher , Norman J. Haughey , Eric O. Freed Proceedings Of The National Academy Of Sciences 120 DOI: 10.1073/pnas.2219475120 Diamond Proposal Number(s): [29812] Show Abstract ▼ Abstract: HIV-1 assembly occurs at the inner leaflet of the plasma membrane (PM) in highly ordered membrane microdomains. The size and stability of membrane microdomains is regulated by activity of the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) that is localized primarily to the inner leaflet of the PM. In this study, we demonstrate that pharmacological inhibition or depletion of nSMase2 in HIV-1-producer cells results in a block in the processing of the major viral structural polyprotein Gag and the production of morphologically aberrant, immature HIV-1 particles with severely impaired infectivity. We find that disruption of nSMase2 also severely inhibits the maturation and infectivity of other primate lentiviruses HIV-2 and simian immunodeficiency virus, has a modest or no effect on nonprimate lentiviruses equine infectious anemia virus and feline immunodeficiency virus, and has no effect on the gammaretrovirus murine leukemia virus. These studies demonstrate a key role for nSMase2 in HIV-1 particle morphogenesis and maturation.	Jul 2023
I04-1-Macromolecular Crystallography (fixed wavelength) Krios II-Titan Krios II at Diamond	Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease Andre Schutzer Godoy , Aline Minalli Nakamura , Alice Douangamath , Yun Song , Gabriela Dias Noske , Victor Oliveira Gawriljuk , Rafaela Sachetto Fernandes , Humberto D'Muniz Pereira , Ketllyn irene Zagato Oliveira , Daren Fearon , Alexandre Dias , Tobias Krojer , Michael Fairhead , Alisa Powell , Louise Dunnett , Jose Brandao-Neto , Rachael Skyner , Rod Chalk , Dávid Bajusz , Miklós Bege , Anikó Borbás , György Miklós Keserű , Frank Von Delft , Glaucius Oliva Nucleic Acids Research 579 DOI: 10.1093/nar/gkad314 Diamond Proposal Number(s): [27083, 27023] Open Access Show Abstract ▼ Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.	Apr 2023
Krios II-Titan Krios II at Diamond	An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition Gabriela Dias Noske , Yun Song , Rafaela Sachetto Fernandes , Rod Chalk , Haitem Elmassoudi , Lizbe Koekemoer , C. David Owen , Tarick J. El-Baba , Carol V. Robinson , Glaucius Oliva , Andre Schutzer Godoy Nature Communications 14 DOI: 10.1038/s41467-023-37035-5 Diamond Proposal Number(s): [27083, 29349] Open Access Show Abstract ▼ Abstract: The main protease from SARS-CoV-2 (Mpro) is responsible for cleavage of the viral polyprotein. Mpro self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use C145S Mpro to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis shows that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical for dimerization. A 3.5 Å cryo-EM structure provides details of Mpro N-terminal cleavage outside the constrains of crystal environment. We show that different classes of inhibitors shift the balance between oligomeric states. While non-covalent inhibitor MAT-POS-e194df51-1 prevents dimerization, the covalent inhibitor nirmatrelvir induces the conversion of monomers into dimers, even with intact N-termini. Our data indicates that the Mpro dimerization is triggered by induced fit due to covalent linkage during substrate processing rather than the N-terminal processing.	Mar 2023
Krios I-Titan Krios I at Diamond Krios II-Titan Krios II at Diamond	Structure and activity of particulate methane monooxygenase arrays in methanotrophs Yanan Zhu , Christopher W. Koo , C. Keith Cassidy , Matthew C. Spink , Tao Ni , Laura C. Zanetti-Domingues , Benji Bateman , Marisa Martin-Fernandez , Juan Shen , Yuewen Sheng , Yun Song , Zhengyi Yang , Amy C. Rosenzweig , Peijun Zhang Nature Communications 13 DOI: 10.1038/s41467-022-32752-9 Diamond Proposal Number(s): [21004, 29812] Open Access Show Abstract ▼ Abstract: Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.	Sep 2022
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	The antigenic anatomy of SARS-CoV-2 receptor binding domain Wanwisa Dejnirattisai , Daming Zhou , Helen M. Ginn , Helen M. E. Duyvesteyn , Piyada Supasa , James Brett Case , Yuguang Zhao , Thomas Walter , Alexander J. Mentzer , Chang Liu , Beibei Wang , Guido C. Paesen , Jose Slon-Campos , César López-Camacho , Natasha M. Kafai , Adam L. Bailey , Rita E. Chen , Baoling Ying , Craig Thompson , Jai Bolton , Alex Fyfe , Sunetra Gupta , Tiong Kit Tan , Javier Gilbert-Jaramillo , William James , Michael Knight , Miles W. Carroll , Donal Skelly , Christina Dold , Yanchun Peng , Robert Levin , Tao Dong , Andrew J. Pollard , Julian C. Knight , Paul Klenerman , Nigel Temperton , David R. Hall , Mark A. Williams , Neil G. Paterson , Felicity Bertram , C. Alistair Siebert , Daniel K. Clare , Andrew Howe , Julika Radecke , Yun Song , Alain R. Townsend , Kuan-Ying A. Huang , Elizabeth E. Fry , Juthathip Mongkolsapaya , Michael S. Diamond , Jingshan Ren , David I. Stuart , Gavin R. Screaton Cell DOI: 10.1016/j.cell.2021.02.032 Diamond Proposal Number(s): [27009, 26983] Open Access Show Abstract ▼ Abstract: Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike, and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50<0.1μg/ml) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryo-electron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.	Feb 2021

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