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Matthew R.
Singer
,
Zhen
Li
,
Juan S.
Rey
,
Joshua
Hope
,
Florian
Chenavier
,
Nicola J.
Cook
,
Emma
Punch
,
Jamie
Smith
,
Zhiyu
Zhou
,
Sarah
Maslen
,
Laura
Masino
,
Andrea
Nans
,
Mark
Skehel
,
Ian A.
Taylor
,
Giulia
Zanetti
,
Peijun
Zhang
,
Juan R.
Perilla
,
Alan N.
Engelman
,
Peter
Cherepanov
Open Access
Abstract: HIV-1 integrase (IN) promotes encapsulation of viral genomic RNA into mature viral cores, and this function is a target for ongoing antiretroviral drug development efforts1,2,3. Here we determined the cryogenic electron microscopy (cryo-EM) structure of a primate lentiviral IN in a complex with RNA, revealing a linear filament made of IN octamer repeat units, each comprising a pair of asymmetric homotetramers. The assembly is stabilized through IN–RNA interactions involving mainly the IN C-terminal domains and RNA backbone. The spacing and orientation of the IN filament repeat units closely matched those of consecutive capsid (CA) hexamers within the mature CA lattice. Using cryo-EM images of native purified HIV-1 cores, we refined the structure of the IN filament as it propagates along the luminal side of the CA lattice. Each IN tetramer within the filament nestled in a CA hexamer, engaging closely with the major homology regions. Substitutions of residues involved in IN–CA contacts yielded eccentric virions with RNA nucleoids located outside of the cores. Collectively, our results establish the structural basis for the HIV-1 IN–RNA interaction and reveal that IN forms an RNA-binding module on the luminal side of the mature CA lattice.
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Feb 2026
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Maurizio
Toft
,
Maëva
Meynier
,
Hélène Lubrano
Di Scampamorte
,
Cedric
Vallee
,
Miguel
Salinas
,
Peijun
Zhang
,
Jessica
Tacco
,
Anne-Sophie
Gay
,
Emmanuel
Bourinet
,
Eric
Lingueglia
,
Emmanuel
Deval
Open Access
Abstract: Acid-sensing ion channels (ASICs) are members of the DEG/ENaC family that includes the only known peptide-gated ion channels. While ASICs are gated by protons, they are also sensitive to peptides and are modulated by the molluscan FMRFamide and other mammalian neuropeptides ending by the RFamide motif. We identified a set of synthetic short amidated hexapeptides, which not only end by the RFamide motif but also by CFamide and FCamide, as potent positive modulators of ASIC3 acid-induced activity. We focused on two of them, a RFamide peptide (FR RFamide) and a CFamide peptide (FR Famide), demonstrating that they have similar specificity for and effects on ASIC3. The potentiating effects of the two peptides are due to a strong slow-down of desensitization, leading to an increase in the amount of current induced by acid pH (≤pH6.6), with apparent affinities ranging from 1 to 5 μM. Surprisingly, the washout kinetic of FR RFamide peptide was much slower than those of FR Famide and other known RFamide peptides, suggesting potential differences in their mechanisms of action. Computational modeling and structure-function analysis reveal interactions of both peptides with the non-proton binding site of ASIC3 as already reported before for other RFamide peptides, but our data also suggest possible additional effects of FR RFamide involving directly or indirectly the proton binding domain. These findings expand our understanding of ASICs’ modulation by peptides, identifying novel short modulators of ASIC3, including peptides with new CFamide and FCamide ending motifs, and showing differences between these peptides using their washout kinetic as a new parameter.
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Dec 2025
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Hui
Sun
,
Yanan
Jiang
,
Miaolin
Lan
,
Ming
Zhou
,
Gangshun
Yi
,
Juan
Shen
,
Tingting
Deng
,
Liqin
Liu
,
Yang
Huang
,
Yu
Li
,
Jinfu
Su
,
Yanling
Lin
,
Zhenqin
Chen
,
Lizhi
Zhou
,
Tingting
Li
,
Hai
Yu
,
Tong
Cheng
,
Yali
Zhang
,
Lunzhi
Yuan
,
Shaowei
Li
,
Ying
Gu
,
Peijun
Zhang
,
Ningshao
Xia
,
Qingbing
Zheng
Open Access
Abstract: The rapid evolution of SARS-CoV-2 and the subsequent emergence of Omicron subvariants pose significant challenges to the efficacy of existing vaccines and therapeutics, including those previously reported most broad neutralizing antibodies (bnAbs). Here, we investigated the molecular basis of the altered neutralization profile of a bnAb, 1C4, against recent variants. 1C4 is effective against early variants from Alpha to Omicron BQ.1, but is circumvented by BQ.1.1, XBB and thereafter variants, primarily due to an additional R346T mutation that diminishes its binding affinity. Cryo-electron microscopy analysis revealed that despite the loss of neutralizing potency, 1C4 retained residual binding to the spike protein of immune-evasive variants such as XBB, which harbor altered receptor-binding domain (RBD). Furthermore, 1C4 exhibited a diminished capacity to inhibit ACE2 engagement with Omicron variants, amplifying the intricacies of viral immune evasion tactics. To address this, we employed the mi3-SpyCatcher-based nanoparticle to polymerize 1C4 (mi3-1C4), which reestablished the neutralization potency against recent variants by enhancing avidity via multivalent binding. Such multivalent binding can promote efficient spike aggregation as well as viral cross-linking, thereby providing enhanced protection against both the infection of Beta and XBB variants in a hamster model. Together, our findings delineate the molecular landscape of immune evasion by neutralizing antibodies and provide strategic insight for the adaptation of antibody engineering to keep pace with viral evolution.
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Dec 2025
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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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Open Access
Abstract: As cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) continue to advance, the ability to visualize cellular and organismal structures with unprecedented clarity is redefining the landscape of structural biology. Breakthroughs in imaging technology, sample preparation and image processing now enable the detailed elucidation of cellular architecture, macromolecular organization, and dynamic biological processes at sub-nanometer resolution. Recent methodological advances have propelled the field to new frontiers, facilitating the investigation of complex biological questions across scales—from macromolecular complexes to organism-wide structural insights. This review explores rapidly emerging trends, highlights key innovations that are pushing the boundaries of in situ structural biology, and addresses persistent challenges in expanding the applicability of cryo-EM and cryo-ET across diverse biological systems.
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Aug 2025
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Krios I-Titan Krios I at Diamond
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Gangshun
Yi
,
Dimitrios
Mamalis
,
Mingda
Ye
,
Loic
Carrique
,
Michael
Fairhead
,
Huanyu
Li
,
Katharina L.
Duerr
,
Peijun
Zhang
,
David B.
Sauer
,
Frank
Von Delft
,
Benjamin G.
Davis
,
Robert J. C.
Gilbert
Diamond Proposal Number(s):
[20223, 21004]
Open Access
Abstract: Whilst cryo-electron microscopy(cryo-EM) has become a routine methodology in structural biology, obtaining high-resolution cryo-EM structures of small proteins (<100 kDa) and increasing overall throughput remain challenging. One approach to augment protein size and improve particle alignment involves the use of binding proteins or protein-based scaffolds. However, a given imaging scaffold or linking module may prove inadequate for structure solution and availability of such scaffolds remains limited. Here, we describe a strategy that exploits covalent dimerization of nanobodies to trap an engineered, predisposed nanobody-to-nanobody interface, giving Di-Gembodies as modular constructs created in homomeric and heteromeric forms. By exploiting side-chain-to-side-chain assembly, they can simultaneously display two copies of the same or two distinct proteins through a subunit interface that provides sufficient constraint required for cryo-EM structure determination. We validate this method with multiple soluble and membrane structural targets, down to 14 kDa, demonstrating a flexible and scalable platform for expanded protein structure determination.
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Aug 2025
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Aquilos-CryoFIB at Diamond
Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[29812, 21005]
Open Access
Abstract: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is central to global CO2 fixation. In eukaryotic algae, its catalytic efficiency is enhanced through the pyrenoid - a protein-dense organelle within the chloroplast that concentrates CO2. Although Rubisco structure has been extensively studied in vitro, its native structure, dynamics and interactions within the pyrenoid remain elusive. Here, we present the native Rubisco structure inside the green alga Chlamydomonas reinhardtii determined by cryo-electron tomography and subtomogram averaging of cryo-focused ion beam milled cells. Multiple structural subsets of Rubisco are identified, stochastically distributed throughout the pyrenoid. While Rubisco adopts an active conformation in the best-resolved map, comparison among the subsets reveals significant local variations at the active site, at the large subunit dimer interfaces, and at binding protein contact regions. These findings offer a comprehensive understanding of the structure, dynamics, and functional organization of native Rubisco within the pyrenoid, providing valuable insights into its critical role in CO2 fixation.
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Aug 2025
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Aquilos-CryoFIB at Diamond
Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[29812]
Open Access
Abstract: Direct visualization of HIV-1 nuclear import through the nuclear pore complex (NPC) presents a technical challenge due to the rarity of this process. To enable systematic investigation, we developed a robust in situ system that mimics HIV-1 nuclear import in a near-native context using isolated HIV-1 virus like particles (VLP) cores and permeabilized CD4 + T lymphocyte (CEM) cells. This approach supports docking and translocation of abundant viral cores through nuclear pores into the nucleus. For high-resolution visualization, we implemented an integrated correlative approach to guide cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) imaging, enabling precise targeting and structural characterization of individual nuclear import events. Using this workflow, we visualized 510 HIV-1 VLP cores at distinct stages of nuclear import, capturing key snapshots of the full progression of nuclear import. Subsequent statistical and structural analyses allow classification of core morphologies and identification of translocation-associated remodeling in nuclear pores. This work provides a methodological foundation for dissecting HIV-1 and potentially other viruses nuclear import processes and post-entry events in a controlled and quantitative manner.
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Aug 2025
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Aquilos-CryoFIB at Diamond
Krios III-Titan Krios III at Diamond
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Zhen
Hou
,
Yao
Shen
,
Stanley
Fronik
,
Juan
Shen
,
Jiong
Shi
,
Jialu
Xu
,
Long
Chen
,
Nathan
Hardenbrook
,
Alan N.
Engelman
,
Christopher
Aiken
,
Peijun
Zhang
Diamond Proposal Number(s):
[29812]
Open Access
Abstract: Lentiviruses, such as HIV-1, infect non-dividing cells by traversing the nuclear pore complex (NPC); however, the detailed molecular processes remain unclear. Here we reconstituted functional HIV-1 nuclear import using permeabilized T cells and isolated HIV-1 cores, which significantly increases import events, and developed an integrated three-dimensional cryo-correlative workflow to specifically target and image 1,489 native HIV-1 cores at 4 distinct nuclear import stages using cryo-electron tomography. We found HIV-1 nuclear import depends on both capsid elasticity and nuclear pore adaptability. The NPC acts as a selective filter, preferentially importing smaller cores, while expanding and deforming to accommodate their passage. Brittle mutant cores fail to enter the NPC, while CPSF6-binding-deficient cores enter but stall within the NPC, leading to impaired nuclear import. This study uncovers the interplay between the HIV-1 core and the NPC and provides a framework to dissect HIV-1 nuclear import and downstream events, such as uncoating and integration.
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Jul 2025
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Jul 2025
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