Krios III-Titan Krios III at Diamond
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Yanan
Zhu
,
Alex B.
Kleinpeter
,
Juan S.
Rey
,
Juan
Shen
,
Yao
Shen
,
Jialu
Xu
,
Nathan
Hardenbrook
,
Long
Chen
,
Anka
Lucic
,
Juan R.
Perilla
,
Eric O.
Freed
,
Peijun
Zhang
Diamond Proposal Number(s):
[29812]
Open Access
Abstract: Inositol hexakisphosphate (IP6) promotes HIV-1 assembly by stabilizing the immature Gag lattice and becomes enriched within virions, where it is required for mature capsid assembly. Previously, we identified Gag mutants that package little IP6 yet assemble particles, though they are non-infectious due to defective capsid formation. Here, we report a compensatory mutation, G225R, in the C-terminus of capsid protein (CA) that restores capsid assembly and infectivity in these IP6-deficient mutants. G225R also enhances in vitro assembly of CA into capsid-like particles at far lower IP6 concentrations than required for wild-type CA. CryoEM structures of G225R CA hexamers and lattices at 2.7 Å resolution reveal that the otherwise disordered C-terminus becomes structured, stabilizing hexamer-hexamer interfaces. Molecular dynamics simulations support this mechanism. These findings uncover how HIV-1 can adapt to IP6 deficiency and highlight a previously unrecognized structural role of the CA C-terminus, while offering tools for capsid-related studies.
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Sep 2025
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[29812]
Open Access
Abstract: During HIV-1 maturation, the matrix (MA) lattice underlying the viral membrane undergoes a structural rearrangement, and the newly released capsid (CA) protein forms a mature CA. While it is well established that CA formation is essential for particle infectivity, the functional role of MA structural maturation remains unclear. Here, we examine maturation of an MA triple mutant, L20K/E73K/A82T, which, despite replicating similarly to wild-type (WT) in some cell lines, exhibits distinct biochemical behaviors that suggest altered MA-MA interactions. Cryo–electron tomography with subtomogram averaging reveals that, although the MA lattice in immature L20K/E73K/A82T virions closely resembles that of the WT, mature L20K/E73K/A82T virions lack a detectable MA lattice. All-atom molecular dynamics simulations suggest that this absence results from destabilized inter-trimer MA interactions in mature L20K/E73K/A82T mutant virions. These findings suggest that an ordered, membrane-associated mature MA lattice is not essential for HIV-1 infectivity, providing insights into the structural requirements for HIV-1 particle maturation and generation of infectious particles.
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May 2025
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I03-Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
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James
Hillier
,
Yuguang
Zhao
,
Loic
Carrique
,
Tomas
Malinauskas
,
Reinis R.
Ruza
,
Tao-Hsin
Chang
,
Gangshun
Yi
,
Helen M. E.
Duyvesteyn
,
Jing
Yu
,
Weixian
Lu
,
Els
Pardon
,
Jan
Steyaert
,
Yanan
Zhu
,
Tao
Ni
,
E. Yvonne
Jones
Diamond Proposal Number(s):
[19946, 28713]
Open Access
Abstract: The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating β-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.
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Aug 2024
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[29812]
Open Access
Abstract: The structure of chromatin plays pivotal roles in regulating gene transcription, DNA replication and repair, and chromosome segregation. This structure, however, remains elusive. Here, using cryo-FIB and cryo-ET, we delineate the 3D architecture of native chromatin fibres in intact interphase human T-lymphoblasts and determine the in situ structures of nucleosomes in different conformations. These chromatin fibres are not structured as uniform 30 nm one-start or two-start filaments but are composed of relaxed, variable zigzag organizations of nucleosomes connected by straight linker DNA. Nucleosomes with little H1 and linker DNA density are distributed randomly without any spatial preference. This work will inspire future high-resolution investigations on native chromatin structures in situ at both a single-nucleosome level and a population level under many different cellular conditions in health and disease.
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Oct 2023
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Krios I-Titan Krios I at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Tao
Ni
,
Luiza
Mendonca
,
Yanan
Zhu
,
Andrew
Howe
,
Julika
Radecke
,
Pranav M.
Shah
,
Yuewen
Sheng
,
Anna-Sophia
Krebs
,
Helen M. E.
Duyvesteyn
,
Elizabeth
Allen
,
Teresa
Lambe
,
Cameron
Bisset
,
Alexandra
Spencer
,
Susan
Morris
,
David I.
Stuart
,
Sarah
Gilbert
,
Peijun
Zhang
Diamond Proposal Number(s):
[26987]
Open Access
Abstract: Vaccines against SARS-CoV-2 have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1 vectored HexaPro stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild-type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.
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Sep 2023
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Tao
Ni
,
Qiuyao
Jiang
,
Pei Cing
Ng
,
Juan
Shen
,
Hao
Dou
,
Yanan
Zhu
,
Julika
Radecke
,
Gregory F.
Dykes
,
Fang
Huang
,
Lu-Ning
Liu
,
Peijun
Zhang
Diamond Proposal Number(s):
[29812, 28713]
Open Access
Abstract: Carboxysomes are a paradigm of self-assembling proteinaceous organelles found in nature, offering compartmentalisation of enzymes and pathways to enhance carbon fixation. In α-carboxysomes, the disordered linker protein CsoS2 plays an essential role in carboxysome assembly and Rubisco encapsulation. Its mechanism of action, however, is not fully understood. Here we synthetically engineer α-carboxysome shells using minimal shell components and determine cryoEM structures of these to decipher the principle of shell assembly and encapsulation. The structures reveal that the intrinsically disordered CsoS2 C-terminus is well-structured and acts as a universal “molecular thread” stitching through multiple shell protein interfaces. We further uncover in CsoS2 a highly conserved repetitive key interaction motif, [IV]TG, which is critical to the shell assembly and architecture. Our study provides a general mechanism for the CsoS2-governed carboxysome shell assembly and cargo encapsulation and further advances synthetic engineering of carboxysomes for diverse biotechnological applications.
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Sep 2023
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Krios I-Titan Krios I at Diamond
Krios IV-Titan Krios IV at Diamond
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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
Diamond Proposal Number(s):
[29812]
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.
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Jul 2023
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[29812]
Abstract: The assembly of an HIV-1 particle begins with the construction of a spherical lattice composed of hexamer subunits of the Gag polyprotein. The cellular metabolite inositol hexakisphosphate (IP6) binds and stabilizes the immature Gag lattice via an interaction with the six-helix bundle (6HB), a crucial structural feature of Gag hexamers that modulates both virus assembly and infectivity. The 6HB must be stable enough to promote immature Gag lattice formation, but also flexible enough to be accessible to the viral protease, which cleaves the 6HB during particle maturation. 6HB cleavage liberates the capsid (CA) domain of Gag from the adjacent spacer peptide 1 (SP1) and IP6 from its binding site. This pool of IP6 molecules then promotes the assembly of CA into the mature conical capsid that is required for infection. Depletion of IP6 in virus-producer cells results in severe defects in assembly and infectivity of wild-type (WT) virions. Here we show that in an SP1 double mutant (M4L/T8I) with a hyperstable 6HB, IP6 can block virion infectivity by preventing CA-SP1 processing. Thus, depletion of IP6 in virus-producer cells markedly increases M4L/T8I CA-SP1 processing and infectivity. We also show that the introduction of the M4L/T8I mutations partially rescues the assembly and infectivity defects induced by IP6 depletion on WT virions, likely by increasing the affinity of the immature lattice for limiting IP6. These findings reinforce the importance of the 6HB in virus assembly, maturation, and infection and highlight the ability of IP6 to modulate 6HB stability.
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Jun 2023
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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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