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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Diamond Proposal Number(s):
[29812, 21005]
Open Access
Abstract: RNA polymerase II (RNAPII) drives gene expression through iterative nucleotide addition cycles (NACs) comprising translocation, substrate binding, and catalysis. The lack of pre-catalysis and post-catalysis intermediates has precluded a complete mechanistic understanding of the NAC. Here we present 31 Cryo-electron Microscopy structures (with 43 maps) capturing distinct stages of Saccharomyces cerevisiae RNAPII elongation complex (EC) NAC, including previously intractable transition intermediates. We establish a continuous spectrum of RNAPII EC structural dynamics during the NAC, which can be divided into two coordinated phases: a substrate-induced EC tightening phase and a post-catalysis EC relaxation phase. For the substrate-induced EC tightening phase, the substrate binding initiates allosteric conformational changes across the entire RNAPII EC, including Trigger Loop folding, funnel closure, clamp closure, transcription bubble ordering, and precise alignment of the RNA 3′-end with substrate to form a catalysis-competent configuration. For the post-catalysis EC relaxation phase, we capture the long-sought, short-lived post-catalysis product state and identify a series of intermediates that reveal a reverse conformational transition that facilitates rapid translocation. Together, our findings define a comprehensive structural and dynamic framework for RNAPII NAC, yielding a “molecular movie” of RNAPII in action and revealing a fundamental principle by which the enzyme balances speed and fidelity through coordinated conformational dynamics.
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Jul 2026
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Krios I-Titan Krios I at Diamond
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Yu
Zhang
,
Lijie
Zhong
,
Yun
Song
,
Mingcheng
Guo
,
Keli
Ren
,
Tingting
Yang
,
Yixin
Huang
,
Ilia
Sirotkin
,
Gangshun
Yi
,
Fang
Jiao
,
Peijun
Zhang
,
Robert J. C.
Gilbert
,
Tao
Ni
,
Xiulian
Yu
Diamond Proposal Number(s):
[21004, 29812]
Open Access
Abstract: Malaria-causing Plasmodium parasites must pass through several host cell types to complete their life cycle. This cell traversal is facilitated by perforin-like proteins (PLPs), among which PLP2 is essential for erythrocyte rupture by gametocytes. However, the mechanism by which PLP2 forms pores is not yet understood. Here, we combine cryo-electron microscopy and tomography to reveal the structural basis of Plasmodium vivax PLP2-mediated membrane attack. PvPLP2 assembles on lipid bilayers into heterogeneous arc- and ring-shaped pores with variable stoichiometries. Among them, we determine the structure of a 17-subunit pore complex in which the pore-forming MACPF domains form the central β-barrel, while the peripheral Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains anchor the complex to the membrane surface. A disulfide-stabilized mutant captures an intermediate pre-pore complex prior to membrane insertion, delineating the structural transitions that underpin β-barrel deployment. Functionally, PvPLP2 acts preferentially on the inner leaflet of the erythrocyte membrane, a specificity driven by its affinity for negatively charged lipids. Together, these findings establish the pore-formation pathway for a key Plasmodium virulence factor and provide a structural framework for rational design of transmission-blocking agents that prevent gametocyte egress.
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Jul 2026
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Open Access
Abstract: We reexamine major membrane models against current evidence. Each addresses important questions, but not all are consistent with the available data. Seeking coherence, we propose the strong proteolipid code, a framework of versioned submodels building on an earlier proposal that clarified the relationship between protein and lipid distributions. The first of these submodels introduces glue-field duality as a structure for lipid-mediated protein interactions, classifies protein islands, and bounds autonomous lipid behavior.
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Jul 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[21005]
Open Access
Abstract: The dynamic nature of protein and macromolecular complexes means that the capture of multiple sequential states along a reaction pathway can provide much greater insight into function than that obtained from a single static structure. We present a set of modular, easy-to-implement tools and workflows for optical excitation, on-grid characterization and tightly coupled rapid vitrification, establishing a proof-of-principle framework for time-resolved cryoEM and cryo-electron tomography (cryoET). We apply this framework to E. coli chemotaxis, in which serine-sensitive chemoreceptors initiate signalling upon ligand binding and undergo critical conformational changes within the chemosensory arrays. Using DMNB-caged serine [O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine] as a model trigger, we quantified its photophysical properties and uncaging efficiency using UV–Vis spectroscopy and two-dimensional gas chromatography mass spectrometry (GC×GC-MS). Coupling a femtosecond-pulsed laser to a Vitrobot enabled reproducible reaction-to-vitrification delays of ∼150 ms, yielding intact E. coli minicells with well-preserved chemotaxis arrays suitable for in situ structural analysis by cryoET. This integrated approach provides a robust and generalisable framework for millisecond time-resolved cryoET, laying the groundwork for capturing transient conformational states in their native cellular context.
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Jul 2026
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Open Access
Abstract: Respiratory syncytial virus (RSV) remains a leading cause of severe lower respiratory tract disease in infants, older adults, and immunocompromised individuals. Over the past decade, advances in structural biology, particularly cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), have transformed our understanding of RSV architecture, dynamics, and the mechanisms of entry and replication. High-resolution structures of the prefusion F glycoprotein (pre-F) and its complexes with neutralizing antibodies established the rationale for structure-guided antigen stabilization and directly enabled the development of the first licensed RSV vaccines. Complementary structures of the ribonucleoprotein, polymerase complex, and matrix lattice have broadened therapeutic targets beyond F. Here, we summarize these structural advances; review current structure-guided vaccine, antibody, and antiviral development efforts; and highlight priorities for next-generation vaccines and therapeutics.
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May 2026
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Abstract: The nuclear envelope protects the host genome, yet many viruses must breach this barrier to access nuclear replication machinery. The nuclear pore complex (NPC), the sole gateway for nucleocytoplasmic transport, is therefore a central target for viral nuclear entry. For decades, the mechanisms by which large viral genomes and subviral assemblies traverse this selective channel remained unclear. This was due to limited accessibility to intact nuclear pores, the NPC's massive architecture, and the transient nature of viral nuclear entry. The advent of cryo–electron microscopy (cryo-EM) and cryo–electron tomography (cryo-ET) has transformed this landscape, enabling visualization of NPC architecture and virus-NPC interactions at unprecedented detail. This review summarizes structural insights into NPC architecture and the strategies viruses employ to enter the nucleus. We examine how viruses engage canonical import pathways involving importins and phenylalanine-glycine-nucleoporins, as well as noncanonical mechanisms by which viral components mimic karyopherin. Finally, we offer perspectives on cryo-EM/cryo-ET capturing viral complexes during the entry process, revealing mechanisms of host-machinery exploitation by viruses.
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May 2026
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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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Diamond Proposal Number(s):
[29812, 34631, 21005]
Open Access
Abstract: RNA polymerase II (RNA Pol II) is central to gene expression, but its catalytic mechanism remains elusive due to the absence of high-resolution structural data. The role of water molecules in RNA Pol II catalysis is unknown. Here, we present 3 high-resolution cryo-electron microscopy structures of active Saccharomyces cerevisiae RNA Pol II elongation complexes in distinct catalytic states: two pre-catalysis states at 1.96 Å and 2.26 Å resolution and a post-catalysis state at 2.33 Å resolution. Each structure contains over 700–1,350 ordered water molecules, many located at functionally critical positions. Comparative analysis shows that these waters play essential roles in proton-transfer steps during RNA Pol II catalysis, facilitating substrate recognition and trigger-loop folding during nucleotide addition. Strikingly, these waters are conserved between prokaryotic and eukaryotic transcription machineries (see Mueller and Darst). These findings provide unprecedented mechanistic insights into RNA Pol II catalysis and reveal vital and evolutionarily conserved roles of water molecules in transcription.
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Apr 2026
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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
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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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