Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[20223, 21004]
Open Access
Abstract: Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a pore-forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre-pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre-assembled complete pre-pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre-pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β-hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre-pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.
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Oct 2022
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
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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
Diamond Proposal Number(s):
[21004, 29812]
Open Access
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.
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Sep 2022
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[21004, 20223, 21005]
Open Access
Abstract: Carboxysomes are a family of bacterial microcompartments in cyanobacteria and chemoautotrophs. They encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase catalyzing carbon fixation inside a proteinaceous shell. How Rubisco complexes pack within the carboxysomes is unknown. Using cryo-electron tomography, we determine the distinct 3D organization of Rubisco inside two distant α-carboxysomes from a marine α-cyanobacterium Cyanobium sp. PCC 7001 where Rubiscos are organized in three concentric layers, and from a chemoautotrophic bacterium Halothiobacillus neapolitanus where they form intertwining spirals. We further resolve the structures of native Rubisco as well as its higher-order assembly at near-atomic resolutions by subtomogram averaging. The structures surprisingly reveal that the authentic intrinsically disordered linker protein CsoS2 interacts with Rubiscos in native carboxysomes but functions distinctively in the two α-carboxysomes. In contrast to the uniform Rubisco-CsoS2 association in the Cyanobium α-carboxysome, CsoS2 binds only to the Rubiscos close to the shell in the Halo α-carboxysome. Our findings provide critical knowledge of the assembly principles of α-carboxysomes, which may aid in the rational design and repurposing of carboxysome structures for new functions.
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Jul 2022
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Perforin-like proteins (PLPs) play key roles in mechanisms associated with parasitic disease caused by apicomplexans parasites Plasmodium and Toxoplasma. The T. gondii PLP1 (TgPLP1) mediates tachyzoite egress from cells, while the five Plasmodium PLPs carry out various roles in the life cycle of the parasite and with respect to the molecular basis of disease. Here we focus on Plasmodium vivax PLP1 and PLP2 (PvPLP1 and PvPLP2) compared to TgPLP1. Determination of the crystal structure of the membrane-binding APCβ domain of PvPLP1 reveals notable differences with TgPLP1, reflected in its inability to bind lipid bilayers as TgPLP1 and PvPLP2 do. Molecular dynamics simulations combined with site-directed mutagenesis and functional assays allow dissection of the binding interactions of TgPLP1 and PvPLP2 on lipid bilayers, and reveal similar tropisms for lipids enriched in the inner leaflet of the mammalian plasma membrane. In addition PvPLP2 displays a secondary synergistic interaction side-on from its principal bilayer interface. This study underlines the substantial differences between the biophysical properties of the APCβ domains of apicomplexan PLPs, which reflect their significant sequence diversity. Such differences will be important factors in determining the cell targeting and membrane-binding activity of the different proteins in parasitic life cycles and disease.
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May 2022
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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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