Krios I-Titan Krios I at Diamond
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Carys
Williams
,
Laura M.
Nocka
,
George
Hedger
,
Pragya
Parashara
,
Els
Pardon
,
Naomi R.
Latorraca
,
Ganesh V.
Pusapati
,
Parijat
Sarkar
,
Dorothy
Lartey
,
Lei
Gao
,
Ljiljana
Milenkovic
,
Rod
Chalk
,
Jan
Steyaert
,
Susan
Marqusee
,
Loic
Carrique
,
J. Fernando
Bazan
,
Sarah L.
Rouse
,
Jennifer H.
Kong
,
Christian
Siebold
,
Rajat
Rohatgi
Diamond Proposal Number(s):
[28713]
Open Access
Abstract: Receptor-type E3 ubiquitin ligases enable extracellular signals to control ubiquitylation in the cytoplasm, playing widespread roles in development, metabolism, and immunity. Using cryoelectron microscopy, integrated with biophysical and functional studies, we visualized a human E3 complex composed of two transmembrane proteins, MEGF8 and MOSMO, and the intracellular RING-family protein MGRN1. This MEGF8-MOSMO-MGRN1 (MMM) complex attenuates Hedgehog signaling by ubiquitylating Smoothened (SMO), a G-protein-coupled receptor (GPCR) that transduces morphogen signals. A long helix in the MMM complex engages SMO using an intramembrane degron and extends into the cytoplasm to suspend an activated and precisely oriented RING domain below the plasma membrane. This architecture enables ubiquitylation of the cytoplasmic surface of SMO, reducing SMO abundance at primary cilia. Our structure provides insights into MEGF8 mutations, which cause multi-organ birth defects, and defines a paradigm for how transmembrane E3 ligases control the cell surface abundance of GPCRs and other signaling receptors.
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Jun 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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I09-Surface and Interface Structural Analysis
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Muhammad
Ans
,
Eleni
Fiamegkou
,
Ashok S.
Menon
,
Gaurav C.
Pandey
,
Gaolo J.
Paez Fajardo
,
Harry
Gillions
,
Paolo
Melgari
,
Calum
Clenahan
,
Satish
Bolloju
,
Pardeep K.
Thakur
,
Tien-Lin
Lee
,
Serena A.
Cussen
,
Beth I. J.
Johnston
,
Louis F. J.
Piper
Diamond Proposal Number(s):
[38340]
Open Access
Abstract: Lithium nickel oxide (LNO) cathodes offer high capacity for high-energy-density applications but suffer rapid degradation above 4.2 V due to surface and bulk instabilities. Here, we apply an ultrathin aluminum oxide coating using powder atomic layer deposition to improve surface stability. Pouch cell testing shows that coated LNO delivers improved cycling behavior, retaining 91.2% capacity after 100 cycles at C/3. Operando X-ray diffraction reveals that after aging, coated LNO undergoes a less kinetically hindered delithiation, indicating that the surface coating further provides a surface-to-bulk stabilization effect. Postmortem surface sensitive spectroscopy confirms that the aluminum oxide layer (1) scavenges hydrofluoric acid and (2) suppresses surface reconstruction, reducing impedance growth and improving the surface integrity. Overall, the results demonstrate that ultrathin aluminum oxide coatings effectively mitigate interfacial degradation and enhance bulk electrochemical kinetics, providing an effective and scalable approach toward improving the long-term performance of ultra-Ni-rich cathodes.
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Mar 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[36134]
Open Access
Abstract: In contrast to living organisms, viruses were long thought to lack protein synthesis machinery and instead depend on host factors to translate viral transcripts. Here, we discover that giant DNA viruses encode a distinct and functional IF4F translation-initiation complex to drive protein synthesis, thereby blurring the line between cellular and acellular biology. During infection, eukaryotic IF4F on host ribosomes is replaced by an essential viral IF4F that regulates viral translation, virion formation, and replication plasticity during altered host states. Structural dissection of viral IF4F reveals that the mRNA cap-binding subunit mediates exclusive interactions with viral mRNAs, constituting a molecular switch from translating host to viral proteins. Thus, our study establishes that viruses express a eukaryotic translation-initiation complex for protein synthesis, illuminating a series of evolutionary innovations in a core process of life.
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Mar 2026
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[33230, 39228]
Open Access
Abstract: Adeno-associated viruses (AAVs) are parvoviruses utilized as gene therapy vectors. However, the AAV packaging mechanism is unresolved at the molecular level, creating a bottleneck for vector manufacturing, safety, and efficacy. Here, cryo-EM structures of the Rep helicase packaging motor in complex with the packaging marker DNA (ITR) and the Rep-AAV8 capsid complex are presented. Rep-ITR complexes reveal dynamic oligomeric states on the DNA, elucidating the strand separation mechanism coupled to its ATPase cycle. We observe Rep preferentially bound to empty capsids, with a binding interface likely conserved across the virus family. This complex also unveils a cryptic capsid ATP-binding site which, alongside Rep binding, triggers structural rearrangements priming the capsid for packaging. Collectively, these findings advance the understanding of Rep-mediated packaging, with significant implications for parvovirus virology and viral vector design.
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Mar 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[36097]
Open Access
Abstract: The cavity-creating p53 cancer mutation Y220C, which accounts for an estimated 125,000 new cancer cases per year, serves as an excellent paradigm for the development of mutant p53 reactivators. Several molecules that reactivate this thermolabile cancer mutant by targeting the mutation-induced crevice have been developed, and one of them, rezatapopt, is currently in clinical trials. The less frequently occurring Y220N and Y220S mutations are even more destabilizing than Y220C but create a similar surface crevice, raising the question of whether cancer patients with these mutations might also benefit from rezatapopt treatment. Here, we show that rezatapopt also binds to the Y220N and Y220S mutants, with nanomolar affinity, resulting in a full recovery of wild-type-like stability for the latter. High-resolution crystal structures of all three mutants bound to rezatapopt revealed a conserved binding mode, highlighting key interactions, including multipolar interactions of a fluorine substituent at a chiral center with the protein backbone. Consistent with the biophysical and structural data, rezatapopt reactivated p53 signaling in both Y220C and Y220S mutant cells by restoring the folded conformation and transcriptional activity, leading to anti-proliferative effects and apoptosis, albeit requiring higher compound concentrations in Y220S cells. The Y220N mutant, despite exhibiting high-nanomolar affinity for rezatapopt and substantial stabilization, did not show noticeable effects in cells at the concentrations tested, as rezatapopt binding resulted in only partial compensation for the mutation-induced loss of stability, for which we provide a structural explanation. Our data suggest that the development of clinical pan-Y220C/N/S reactivators, which could benefit an additional 10,000 patients per year, is challenging but not impossible.
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Feb 2026
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Double-stranded (ds)RNA viruses replicate and transcribe their genome within a proteinaceous viral capsid to evade host cell defenses. While Reovirales members use conservative transcription, most dsRNA viruses, including cystoviruses, utilize semi-conservative transcription, in which a newly synthesized positive strand replaces the parental positive strand, which is released as mRNA. Here, we visualize semi-conservative transcription activation in cystovirus ɸ6 double-layered particles using cryogenic electron microscopy. We observe nucleotide-triggered disassembly of the domain-swapped outer capsid layer, subsequent expansion of the inner capsid layer, and stepwise assembly of transcription complexes at the opposing poles of the spooled dsRNA genome. These complexes consist of the viral polymerases embedded into a triskelion formed by the minor protein P7, which we show as essential for continuous transcription. The packaging hexamers proximal to the transcription sites channel the viral mRNA exit. Our results define the complex molecular pathway from the quiescent state to activated semi-conservative transcription.
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Jan 2026
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B21-High Throughput SAXS
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Sung Ryul
Choi
,
Thorsten B.
Blum
,
Matteo
Giono
,
Bibhas
Roy
,
Ioannis
Vakonakis
,
Dominic
Schmid
,
Nicole
Oelgarth
,
Apisha
Ranganathan
,
Alvar D.
Gossert
,
G. V.
Shivashankar
,
Alfred
Zippelius
,
Michel O.
Steinmetz
Open Access
Abstract: Microtubules have long been recognized as upstream mediators of intracellular signaling, but the mechanisms underlying this fundamental function remain elusive. Here, we identify the structural basis by which microtubules regulate the guanine nucleotide exchange factor H1 (GEFH1), a key activator of the Ras homolog family member A (RhoA) pathway. We show that specific features of the microtubule lattice bind the C1 domain of GEFH1, leading to the sequestration and inactivation of this signaling protein. Targeted mutations in C1 residues disrupt this interaction, triggering GEFH1 release and activation of RhoA-dependent immune responses. Building on this sequestration-and-release mechanism, we identify microtubule-binding C1 domains in additional signaling proteins, including other guanine nucleotide exchange factors (GEFs), kinases, a GTPase-activating protein (GAP), and a tumor suppressor, and show that microtubule-mediated regulation via C1 domains is conserved in the Ras association domain-containing protein 1A (RASSF1A). Our findings establish a structural framework for understanding how microtubules can function as spatiotemporal signal sensors, integrating and processing diverse signaling pathways to control important cellular processes.
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Dec 2025
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Aquilos-CryoFIB at Diamond
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Diamond Proposal Number(s):
[31336]
Open Access
Abstract: Regulation of Lamin A/C levels and distribution is crucial for nuclear integrity and mechanotransduction via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Dysregulation of Lamin A/C correlates with poor cancer prognosis, and its levels determine sensitivity to the microtubule-stabilising drug paclitaxel. Paclitaxel is well-known for disrupting mitosis, yet it also reduces tumour size in slow-dividing tumours, indicating an additional, poorly characterised interphase mechanism.
Here, we reveal that paclitaxel induces nuclear aberrations in interphase through SUN2-dependent Lamin A/C disruption. Using advanced optical imaging and electron cryo-tomography, we show the formation of aberrant microtubule-vimentin bundles during paclitaxel treatment, which coincides with nuclear deformation and altered Lamin A/C protein levels and organisation at the nuclear envelope. SUN2 is required for Lamin A/C reduction in paclitaxel and is in turn regulated by polyubiquitination. Furthermore, Lamin A/C expression levels determine not only cell survival during treatment but also recovery after drug removal.
Our findings support a model in which paclitaxel acts through both defective mitosis and interphase nuclear-cytoskeletal disruption, providing additional mechanistic insights into a widely used anticancer drug.
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Dec 2025
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I04-Macromolecular Crystallography
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Open Access
Abstract: Integrin-mediated cell–matrix adhesions regulate communication between cells and the extracellular matrix. In matrix-secreting cells, fibrillar adhesions (FBs) containing high levels of α5β1 integrins and the tensin3 adaptor protein are essential for fibronectin (FN) fibrillogenesis. Here, we demonstrate that tensin3 binds to four helical regions (R3, R4, R8, and R11) of talin, the principal integrin activator. Structural analysis revealed the residues critical for the tensin3–talin interaction, and mutational analysis showed that talin R8 and R11 are essential for FB formation and FN fibrillogenesis. Cellular experiments demonstrate that tensin3 binding to talin not only regulates integrin activation, but also modulates tensin3’s propensity to undergo liquid–liquid phase separation (LLPS). Formation of such LLPS condensates increased when cells were plated on soft substrates compared with stiff ones. This effect was abolished by blocking the interaction between tensin3 and talin. Our data suggest a model in which LLPS condensates provide a signaling platform involved in cellular responses to sudden changes in tissue mechanics.
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Nov 2025
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