Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[26464]
Abstract: Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.
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Jan 2022
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Tao
Ni
,
Yanan
Zhu
,
Zhengyi
Yang
,
Chaoyi
Xu
,
Yuriy
Chaban
,
Tanya
Nesterova
,
Jiying
Ning
,
Till
Böcking
,
Michael W.
Parker
,
Christina
Monnie
,
Jinwoo
Ahn
,
Juan R.
Perilla
,
Peijun
Zhang
Diamond Proposal Number(s):
[21004, 20223]
Open Access
Abstract: The viral capsid plays essential roles in HIV replication and is a major platform engaging host factors. To overcome challenges in study native capsid structure, we used the perfringolysin O to perforate the membrane of HIV-1 particles, thus allowing host proteins and small molecules to access the native capsid while improving cryo–electron microscopy image quality. Using cryo–electron tomography and subtomogram averaging, we determined the structures of native capsomers in the presence and absence of inositol hexakisphosphate (IP6) and cyclophilin A and constructed an all-atom model of a complete HIV-1 capsid. Our structures reveal two IP6 binding sites and modes of cyclophilin A interactions. Free energy calculations substantiate the two binding sites at R18 and K25 and further show a prohibitive energy barrier for IP6 to pass through the pentamer. Our results demonstrate that perfringolysin O perforation is a valuable tool for structural analyses of enveloped virus capsids and interactions with host cell factors.
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Nov 2021
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B24-Cryo Soft X-ray Tomography
Krios II-Titan Krios II at Diamond
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Luiza
Mendonca
,
Andrew
Howe
,
James B.
Gilchrist
,
Yuewen
Sheng
,
Dapeng
Sun
,
Michael L.
Knight
,
Laura C.
Zanetti-Domingues
,
Benji
Bateman
,
Anna-Sophia
Krebs
,
Long
Chen
,
Julika
Radecke
,
Vivian D.
Li
,
Tao
Ni
,
Ilias
Kounatidis
,
Mohamed A.
Koronfel
,
Marta
Szynkiewicz
,
Maria
Harkiolaki
,
Marisa
Martin-Fernandez
,
William
James
,
Peijun
Zhang
Diamond Proposal Number(s):
[21004, 26987]
Open Access
Abstract: Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events – e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.
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Jul 2021
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Krios II-Titan Krios II at Diamond
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Tuomas
Huokko
,
Tao
Ni
,
Gregory F.
Dykes
,
Deborah M.
Simpson
,
Philip
Brownridge
,
Fabian D.
Conradi
,
Robert J.
Beynon
,
Peter J.
Nixon
,
Conrad W.
Mullineaux
,
Peijun
Zhang
,
Lu-Ning
Liu
Diamond Proposal Number(s):
[21004]
Open Access
Abstract: How thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.
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Jun 2021
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Chuang
Liu
,
Luiza
Mendonca
,
Yang
Yang
,
Yuanzhu
Gao
,
Chenguang
Shen
,
Jiwei
Liu
,
Tao
Ni
,
Bin
Ju
,
Congcong
Liu
,
Xian
Tang
,
Jinli
Wei
,
Xiaomin
Ma
,
Yanan
Zhu
,
Weilong
Liu
,
Shuman
Xu
,
Yingxia
Liu
,
Jing
Yuan
,
Jing
Wu
,
Zheng
Liu
,
Zheng
Zhang
,
Lei
Li
,
Peiyi
Wang
,
Peijun
Zhang
Open Access
Abstract: The ongoing global pandemic of coronavirus disease 2019 (COVID-19) resulted from the outbreak of SARS-CoV-2 in December 2019. Currently, multiple efforts are being made to rapidly develop vaccines and treatments to fight COVID-19. Current vaccine candidates use inactivated SARS-CoV-2 viruses; therefore, it is important to understand the architecture of inactivated SARS-CoV-2. We have genetically and structurally characterized β-propiolactone-inactivated viruses from a propagated and purified clinical strain of SARS-CoV-2. We observed that the virus particles are roughly spherical or moderately pleiomorphic. Although a small fraction of prefusion spikes are found, most spikes appear nail shaped, thus resembling a postfusion state, where the S1 protein of the spike has disassociated from S2. Cryoelectron tomography and subtomogram averaging of these spikes yielded a density map that closely matches the overall structure of the SARS-CoV postfusion spike and its corresponding glycosylation site. Our findings have major implications for SARS-CoV-2 vaccine design, especially those using inactivated viruses.
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Oct 2020
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Krios I-Titan Krios I at Diamond
Krios IV-Titan Krios IV at Diamond
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Tao
Ni
,
Samuel
Gerard
,
Gongpu
Zhao
,
Kyle
Dent
,
Jiying
Ning
,
Jing
Zhou
,
Jiong
Shi
,
Jordan
Anderson-Daniels
,
Wen
Li
,
Sooin
Jang
,
Alan N.
Engelman
,
Christopher
Aiken
,
Peijun
Zhang
Diamond Proposal Number(s):
[14856, 21004]
Abstract: The mature retrovirus capsid consists of a variably curved lattice of capsid protein (CA) hexamers and pentamers. High-resolution structures of the curved assembly, or in complex with host factors, have not been available. By devising cryo-EM methodologies for exceedingly flexible and pleomorphic assemblies, we have determined cryo-EM structures of apo-CA hexamers and in complex with cyclophilin A (CypA) at near-atomic resolutions. The CA hexamers are intrinsically curved, flexible and asymmetric, revealing the capsomere and not the previously touted dimer or trimer interfaces as the key contributor to capsid curvature. CypA recognizes specific geometries of the curved lattice, simultaneously interacting with three CA protomers from adjacent hexamers via two noncanonical interfaces, thus stabilizing the capsid. By determining multiple structures from various helical symmetries, we further revealed the essential plasticity of the CA molecule, which allows formation of continuously curved conical capsids and the mechanism of capsid pattern sensing by CypA.
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Aug 2020
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Yuguang
Zhao
,
Daming
Zhou
,
Tao
Ni
,
Dimple
Karia
,
Abhay
Kotecha
,
Xiangxi
Wang
,
Zihe
Rao
,
E. Yvonne
Jones
,
Elizabeth E.
Fry
,
Jingshan
Ren
,
David I.
Stuart
Open Access
Abstract: Coxsackievirus A10 (CV-A10) is responsible for an escalating number of severe infections in children, but no prophylactics or therapeutics are currently available. KREMEN1 (KRM1) is the entry receptor for the largest receptor-group of hand-foot-and-mouth disease causing viruses, which includes CV-A10. We report here structures of CV-A10 mature virus alone and in complex with KRM1 as well as of the CV-A10 A-particle. The receptor spans the viral canyon with a large footprint on the virus surface. The footprint has some overlap with that seen for the neonatal Fc receptor complexed with enterovirus E6 but is larger and distinct from that of another enterovirus receptor SCARB2. Reduced occupancy of a particle-stabilising pocket factor in the complexed virus and the presence of both unbound and expanded virus particles suggests receptor binding initiates a cascade of conformational changes that produces expanded particles primed for viral uncoating.
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Jan 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Tao
Ni
,
Fang
Jiao
,
Xiulian
Yu
,
Saša
Aden
,
Lucy
Ginger
,
Sophie I.
Williams
,
Fangfang
Bai
,
Vojtech
Prazak
,
Dimple
Karia
,
Phillip
Stansfeld
,
Peijun
Zhang
,
George
Munson
,
Gregor
Anderluh
,
Simon
Scheuring
,
Robert J. C.
Gilbert
Abstract: Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.
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Jan 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Toxoplasma and Plasmodium are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The Toxoplasma gondii PLP1 (TgPLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of TgPLP1. The MACPF domain forms hexameric assemblies, with ring and helix geometries, and the APCβ domain has a novel β-prism fold joined to the MACPF domain by a short linker. Molecular dynamics simulations suggest that the helical MACPF oligomer preserves a biologically important interface, whereas the APCβ domain binds preferentially through a hydrophobic loop to membrane phosphatidylethanolamine, enhanced by the additional presence of inositol phosphate lipids. This mode of membrane binding is supported by site-directed mutagenesis data from a liposome-based assay. Together, these structural and biophysical findings provide insights into the molecular mechanism of membrane targeting by TgPLP1.
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Mar 2018
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain comprising F0-F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association. We present the crystal structure of murine kindlin-3 PH domain determined at 2.23Å resolution and characterise its lipid binding using biophysical and computational approaches. Molecular dynamics (MD) simulations suggest flexibility in the PH domain loops connecting β-strands forming the putative phosphatidylinositol phosphate (PtdInsP) binding site. Simulations with PtdInsP-containing bilayers reveal that the PH domain associates with PtdInsP molecules mainly via the positively charged surface presented by the β1-β2 loop and that it binds with somewhat higher affinity to PtdIns(3,4,5)P3 compared to PtdIns(4,5)P2. Surface plasmon resonance (SPR) with lipid headgroups immobilised and the PH domain as analyte indicate affinities of 300 μM for PtdIns(3,4,5)P3 and 1mM for PtdIns(4,5)P2. In contrast, SPR studies with immobilised PH domain and lipid nanodiscs as analyte show affinities of 0.40 µM for PtdIns(3,4,5)P3 and no affinity for PtdIns(4,5)P2 when the inositol phosphate constitutes 5% of the total lipids (~5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P3 composition to 1% abolishes nanodisc binding to the PH domain, as does site-directed mutagenesis of two lysines within the β1-β2 loop. Binding of PtdIns(3,4,5)P3 by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P3 clustering in the binding of some PH domains and not others, highlighting the importance lipid mobility and clustering for the biophysical assessment of protein-membrane interactions.
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Dec 2016
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