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Abner
Velazco
,
Thomas
Glen
,
Sven
Klumpe
,
Avery
Pennington
,
Jianguo
Zhang
,
Jake L. R.
Smith
,
Calina
Glynn
,
William
Bowles
,
Maryna
Kobylynska
,
Roland A.
Fleck
,
James H.
Naismith
,
Judy S.
Kim
,
Michele C.
Darrow
,
Michael
Grange
,
Angus I.
Kirkland
,
Maud
Dumoux
Open Access
Abstract: Scanning electron microscopy (SEM) of frozen-hydrated biological samples allows imaging of subcellular structures at the mesoscale in a representation of their native state. Combined with focused ion beam milling (FIB), serial FIB/SEM can be used to build a 3-dimensional model of cells and tissues. The correlation of specific regions of interest with cryo-electron microscopy (cryoEM) can additionally enable subsequent high-resolution analysis. However, the use of serial FIB/SEM imaging-based methods is often limited due to charging artefacts arising from insulating areas of cryogenically preserved samples. Here, we demonstrate the use of interleaved scanning to attenuate these artefacts, allowing the observation of biological features that otherwise would be masked or distorted. We apply our method to samples where inherent features were not visible using conventional scanning. These examples include membrane contact sites within mammalian cells, visualisation of the degradation compartment in the algae E. gracilis and observation of a network of membranes within different types of axons in an adult mouse cortex. The proposed alternative scanning method could also be applied to imaging other non-conductive specimens in SEM.
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Jun 2025
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Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[29812]
Open Access
Abstract: SARS-CoV-2 entry into host cells is mediated by the spike protein, which drives membrane fusion. While cryo-EM reveals stable prefusion and postfusion conformations of the spike, the transient fusion intermediate states during the fusion process remain poorly understood. Here, we design a near-native viral fusion system that recapitulates SARS-CoV-2 entry and use cryo-electron tomography (cryo-ET) to capture fusion intermediates leading to complete fusion. The spike protein undergoes extensive structural rearrangements, progressing through extended, partially folded, and fully folded intermediates prior to fusion-pore formation, a process that depends on protease cleavage and is inhibited by the WS6 S2 antibody. Upon interaction with ACE2 receptor dimer, spikes cluster at membrane interfaces and following S2’ cleavage concurrently transition to postfusion conformations encircling the hemifusion and initial fusion pores in a distinct conical arrangement. S2’ cleavage is indispensable for advancing fusion intermediates to the fully folded postfusion state, culminating in membrane integration. Subtomogram averaging reveals that the WS6 S2 antibody binds to the spike’s stem-helix, crosslinks and clusters prefusion spikes, as well as inhibits refolding of fusion intermediates. These findings elucidate the entire process of spike-mediated fusion and SARS-CoV-2 entry, highlighting the neutralizing mechanism of S2-targeting antibodies.
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Jun 2025
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[22006, 30374]
Open Access
Abstract: During synthesis of the ribosomal RNA precursor, RNA polymerase I (Pol I) monitors DNA integrity but its response to DNA damage remains poorly studied. Abasic sites are among the most prevalent DNA lesions in eukaryotic cells, and their detection is critical for cell survival. We report cryo-EM structures of Pol I in different stages of stalling at abasic sites, supported by in vitro transcription studies. Slow nucleotide addition opposite abasic sites occurs through base sandwiching between the RNA 3′-end and the Pol I bridge helix. Templating abasic sites can also cause Pol I cleft opening, which enables the A12 subunit to access the active center. Nucleotide addition opposite the lesion induces a translocation intermediate where DNA bases tilt to form hydrogen bonds with the new RNA base. These findings reveal unique mechanisms of Pol I stalling at abasic sites, differing from arrest by bulky lesions or abasic site handling by RNA polymerase II.
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Jun 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Zihao
Wang
,
Guodong
Cao
,
Miranda P.
Collier
,
Xingyu
Qiu
,
Sophie
Broadway-Stringer
,
Dominik
Šaman
,
Jediael Z. Y.
Ng
,
Navoneel
Sen
,
Amar J.
Azad
,
Charlotte
Hooper
,
Johannes
Zimmermann
,
Michael A.
Mcdonough
,
Jurgen
Brem
,
Patrick
Rabe
,
Haigang
Song
,
T. Reid
Alderson
,
Christopher J.
Schofield
,
Jani R.
Bolla
,
Kristina
Djinovic-Carugo
,
Dieter O.
Fürst
,
Bettina
Warscheid
,
Matteo T.
Degiacomi
,
Timothy M.
Allison
,
Georg K. A.
Hochberg
,
Carol V.
Robinson
,
Katja
Gehmlich
,
Justin L. P.
Benesch
Open Access
Abstract: The biomechanical properties and responses of tissues underpin a variety important of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. FLNC is a multi-functional protein that interacts with a variety of partners, however, how it is regulated at the molecular level is not well understood. Here we investigate its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We find that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we solve by X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work therefore shows, structurally, how HSPB7 acts as a specific molecular chaperone that regulates FLNC dimerisation.
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May 2025
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Krios I-Titan Krios I at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[20287, 26703]
Open Access
Abstract: Bacterial mating, or conjugation, was discovered nearly 80 years ago as a process transferring genes from one bacterial cell (the donor) to another (the recipient). It requires three key multiprotein complexes in the donor cell: a DNA-processing machinery called the relaxosome, a double-membrane spanning type 4 secretion system (T4SS), and an extracellular appendage termed pilus. While the near-atomic resolution structures of the T4SS and pilus are already known, that of the relaxosome has not been reported to date. Here, we describe the cryo-EM structure of the fully assembled relaxosome encoded by the paradigm F plasmid in two different states corresponding to distinct functional steps along the DNA processing reaction. By varying the structures of model DNAs we delineate conformational changes required to initiate conjugation. Mutational studies of the various protein-protein and protein-DNA interaction hubs suggest a complex sensitive to trigger signals, that could arise from cell-to-cell contacts with recipient cells.
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May 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[15916]
Open Access
Abstract: Wnt signal transduction relies on the direct inhibition of GSK3 by phosphorylated PPPSPxS motifs within the cytoplasmic tail of the LRP6 co-receptor. How GSK3 is recruited to LRP6 remains unclear. Here, we use nuclear magnetic resonance spectroscopy to identify the membrane-proximal PPPSPxS motif and its flanking sequences as the primary binding site for both Axin and GSK3, and an intrinsically disordered segment of Axin as its LRP6-interacting region (LIR). Co-immunoprecipitation and CRISPR-engineered mutations in endogenous Axin indicate that its docking at LRP6 is antagonized by a phospho-dependent foldback within LIR and by a PRTxR motif that allows Axin and GSK3 to form a multi-pronged interaction which favors their detachment from LRP6. Crucially, signaling by LRP6 also depends on its binding to the AP2 clathrin adaptor. We propose that the Wnt-driven clustering of LRP6 within clathrin-coated locales allows the Axin-GSK complex to dock at adjacent LRP6 molecules, while also exposing it to co-targeted kinases that change its activity in Wnt signal transduction.
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May 2025
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Open Access
Abstract: ragment screening by crystallography has recently skyrocketed. Multiple synchrotrons have built specialized screening platforms, established workflows, and assembled compound libraries. Crystallographic fragment screening is now widely accessible to groups that had previously not considered the approach. While hundreds of crystallographic fragment-screening campaigns have been conducted in the last few years, most of the underlying data have neither been published nor made publicly accessible. This perspective highlights the importance of establishing effective mechanisms for preserving large and often heterogeneous groups of datasets intrinsic to crystallographic fragment-screening campaigns, thereby ensuring their accessibility for advancing research and enabling applications such as training AI-based models.
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May 2025
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I09-Surface and Interface Structural Analysis
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Connor
Fields
,
Aleksandra
Foerster
,
Sadegh
Ghaderzadeh
,
Ilya
Popov
,
Bang
Huynh
,
Filipe
Junqueira
,
Tyler
James
,
Sofia
Alonso Perez
,
David A.
Duncan
,
Tien-Lin
Lee
,
Yitao
Wang
,
Sally
Bloodworth
,
Gabriela
Hoffman
,
Mark
Walkey
,
Richard J.
Whitby
,
Malcolm H.
Levitt
,
Brian
Kiraly
,
James N.
O'Shea
,
Elena
Besley
,
Philip
Moriarty
Diamond Proposal Number(s):
[31574]
Open Access
Abstract: Charge transfer is fundamentally dependent on the overlap of the orbitals comprising the transport pathway. This has key implications for molecular, nanoscale, and quantum technologies, for which delocalization (and decoherence) rates are essential figures of merit. Here, we apply the core hole clock technique—an energy-domain variant of ultrafast spectroscopy—to probe the delocalization of a photoexcited electron inside a closed molecular cage, namely the Ar 2p54s1 state of Ar@C60. Despite marginal frontier orbital mixing in the ground configuration, almost 80% of the excited state density is found outside the buckyball due to the formation of a markedly diffuse hybrid orbital. Far from isolating the intracage excitation, the surrounding fullerene is instead a remarkably efficient conduit for electron transfer: we measure characteristic delocalization times of 6.6 ± 0.3 fs and ≲ 500 attoseconds, respectively, for a 3D Ar@C60 film and a 2D monolayer on Ag(111).
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May 2025
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I08-Scanning X-ray Microscopy beamline (SXM)
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Rhiannon L.
Jones
,
Jon R.
Hawkings
,
Michael P.
Meredith
,
Maeve C.
Lohan
,
Oliver W.
Moore
,
Robert M.
Sherrell
,
Jessica N.
Fitzsimmons
,
Majid
Kazemian
,
Tohru
Araki
,
Burkhard
Kaulich
,
Amber L.
Annett
Diamond Proposal Number(s):
[30572, 32502]
Open Access
Abstract: Iron is an essential micronutrient for phytoplankton and plays an integral role in the marine carbon cycle. The supply and bioavailability of iron are therefore important modulators of climate over glacial-interglacial cycles. Inputs of iron from the Antarctic continental shelf alleviate iron limitation in the Southern Ocean, driving hotspots of productivity. Glacial meltwater fluxes can deliver high volumes of particulate iron. Here, we show that glacier meltwater provides particles rich in iron(II) to the Antarctic shelf surface ocean. Particulate iron(II) is understood to be more bioavailable to phytoplankton, but less stable in oxic seawater, than iron(III). Using x-ray microscopy, we demonstrate co-occurrence of iron and organic carbon-rich phases, suggesting that organic carbon retards the oxidation of potentially-bioavailable iron(II) in oxic seawater. Accelerating meltwater fluxes may provide an increasingly important source of bioavailable iron(II)-rich particles to the Antarctic surface ocean, with implications for the Southern Ocean carbon pump and ecosystem productivity.
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May 2025
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I03-Macromolecular Crystallography
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Zhixiang
Chen
,
Harshil
Dhruv
,
Xuqing
Zhang
,
Rohan Kalyan
Rej
,
Longchuan
Bai
,
Donna
Mceachern
,
Paul
Kirchhoff
,
Rakesh
Nagilla
,
Larry J.
Jolivette
,
Cory T.
Rice
,
Peter
Orth
,
Corey O.
Strickland
,
E. Scott
Priestley
,
Helai P.
Mohammad
,
Meilin
Wang
,
Bo
Wen
,
Duxin
Sun
,
Zhihua
Sui
,
Shaomeng
Wang
Open Access
Abstract: IKZF2 (Helios) is a transcription factor that is selectively expressed by Tregs and is essential for preserving the function and stability of Tregs in the tumor microenvironment (TME), where it suppresses the anti-tumor immune response. Targeted IKZF2 degradation by small molecules represents a promising strategy for the development of a new class of cancer immunotherapy. Herein, we describe the discovery of PVTX-405, a potent, effective, highly selective, and orally efficacious IKZF2 molecular glue degrader. PVTX-405 degrades IKZF2 (DC50 = 0.7 nM and Dmax = 91%) while sparing other CRBN neo-substrates. Degradation of IKZF2 by PVTX-405 increases production of inflammatory cytokine IL-2 and reduces the suppressive activity of Tregs, leading to an increase in Teff cell proliferation. Once-daily oral administration of PVTX-405 as single agent significantly delays the growth of MC38 tumors in a syngeneic tumor model using humanized CRBN mice. PVTX-405 in combination with anti-PD1 or anti-LAG3 significantly increases animal survival compared to anti-PD1 or anti-LAG3 alone. Together, these results demonstrate that PVTX-405 is a promising IKZF2 degrader for clinical development for the treatment of human cancers.
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May 2025
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