Krios I-Titan Krios I at Diamond
Krios IV-Titan Krios IV at Diamond
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Yasunori
Noguchi
,
Almutasem
Saleh
,
Sarah
Schneider
,
Marina E.
Ivanova
,
Zhuo Angel
Chen
,
Lepakshi
Ranjha
,
Ricardo
Aramayo
,
Silvia
Tognetti
,
Sarah V.
Faull
,
Juri
Rappsilber
,
Christian
Speck
Diamond Proposal Number(s):
[25127, 18659]
Open Access
Abstract: Regulated helicase activation by DDK kinase is central for genome stability. However, how DDK phosphorylation primes the MCM2-7 double hexamer (DH) for Sld3-Sld7 binding and Cdc45 loading remained unclear. We define this mechanism through cryo-EM structures of MCM2-7 DH-Sld3-Sld7 (MS) and MCM2-7 DH-Sld3-Sld7-Cdc45 (MSC). We reveal that the autoinhibitory Mcm4 tail engages not only Mcm4 but also Mcm6. Upon DDK-dependent phosphorylation, both of these sites become accessible. In the context of the MS structure, we identify that two short Sld3 motifs that contact Mcm4 and Mcm6 read out the DH phosphorylation state, while the Sld3 Treslin domain (STD) binds to Mcm2. In the MSC structure, Cdc45 dislodges the Sld3 STD from Mcm2, allowing Sld3 to position Cdc45 at the Mcm2/Mcm5 interface. Mutagenesis of the Sld3 STD-Cdc45 interface disrupts Cdc45 loading, validating this interaction. Together, our data reveal a phosphorylation-encoded mechanism coupling DDK-activated Mcm4/Mcm6 surfaces to distal Cdc45 placement, explaining how firing factors choreograph the DH-to-CMG transition.
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Aug 2026
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[42268, 35335]
Abstract: Central to Alzheimer’s disease pathology are prefibrillar oligomer assemblies of amyloid-β (Aβ) peptide. A widely discussed hypothesis proposes that amyloid-β oligomers insert into neuronal lipid membranes, disrupting their integrity and causing a loss of cellular homeostasis in Alzheimer’s disease. This membrane disruption is believed to be a major source of Aβ-induced neurotoxicity. Cryo electron tomography (cryo-ET) has facilitated 3D nanoscale imaging of Aβ-membrane interactions under near-native conditions. Analyses of small extracellular vesicles (sEVs) reveals that Aβ oligomers including annular and curvilinear extended oligomers (CLEOs) exhibit extensive binding to cell-derived lipid membranes, including insertion into and carpeting of the lipid bilayer. Notably, these oligomeric assemblies were also internalized and concentrated within the cell-derived exosomes and other small sEVs. Enrichment of Aβ oligomers within the vesicles typically ranged between 5 to 20 times the external Aβ levels depending on the vesicle size and curvature. In contrast, monomeric and fibrillar forms of Aβ displayed minimal membrane interaction. Once internalized CLEOs appear to be trapped in an oligomeric form and do not readily go on to form fibrils. Studies with vesicles of brain lipid extract indicate the Aβ internalization does not require the presence of a membrane protein. Our in vitro studies underscore the membrane-disruptive capacity of oligomeric Aβ species and suggest a role of sEVs in concentrating toxic Aβ oligomers and transporting oligomers across the brain interstitium.
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Jul 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, 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
Krios IV-Titan Krios IV at Diamond
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Jose Enrique
Gonzalez-Prada
,
Sulin
Liu
,
Chuhan
Shang
,
Chloe S.
Chernoff
,
Damian P.
Bright
,
Martin
Mortensen
,
Charlotte F.
Jones
,
Stephanie
Nestorow
,
Vikram Babu
Kasaragod
,
Wan-Na
Chen
,
Saad
Hannan
,
Jianchong
Zhou
,
Alexander W. E.
Dunn
,
Asma
Soltani
,
Richard J.
Turner
,
Natasha M.
Duggan
,
Yin
Yuan
,
Ayla A.
Wahid
,
Steven W.
Hardwick
,
Suzanne
Scott
,
Dimitri Y.
Chirgadze
,
Els
Pardon
,
Jan
Steyaert
,
A. Radu
Aricescu
,
Ole
Paulsen
,
David
Belin
,
Trevor G.
Smart
,
Paul S.
Miller
Diamond Proposal Number(s):
[31589, 37678]
Open Access
Abstract: γ-Aminobutyric acid type-A (GABAA) receptors are the principal mediators of inhibitory neurotransmission in the human central nervous system. The α2- and α3-containing subtypes have tightly controlled spatial expression profiles, which influence anxiety, nociception, epilepsy, and autism. α2/α3-Selective small molecules compromise on strength of effect (efficacy) to avoid off-subtype modulation. To break this pharmacological deadlock, we study here a panel of nanobodies (NBs) raised against α2- and α3-containing GABAA receptors. We identify subtype selective silent binders, positive allosteric modulators (PAMs), and inhibitors. Cryo–electron microscopy structures explain the binding modes and molecular mechanisms of action of representative NBs. Modulators exhibit distinct synaptic and extrasynaptic functional profiles in brain slices and neuronal networks and can reduce anxiety in vivo. These selective and efficacious NBs (whether inhibitors or positive modulators) enable strong yet precise pharmacological control of α2/α3-containing subtypes to advance basic research and as potential therapeutic leads to treat neuropsychiatric disorders.
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Jul 2026
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Krios IV-Titan Krios IV at Diamond
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Carlo J. A.
Verhoef
,
Charlotte
Crowe
,
Mark A
Nakasone
,
Aitana
Delacuadra-Basté
,
Tessa
Harzing
,
Naomi A. S.
Span
,
Gajanan
Sathe
,
Laura C.
Demmers
,
Kentaro
Iso
,
Christian
Ottmann
,
Luc
Brunsveld
,
Alessio
Ciulli
,
Peter J.
Cossar
Diamond Proposal Number(s):
[31827]
Open Access
Abstract: Proteins lacking defined ligandable pockets remain challenging drug targets. Here, we develop a molecular glue-based PROTAC (MGPROTAC) approach that chemically conjugates a molecular glue stabilizer to a VHL-recruiting ligand to capture and ubiquitinate the 14-3-3/Estrogen receptor α (ERα) complex. Our designed MGPROTACs engage a composite interface between 14-3-3 and the disordered F-domain of ERα, promoting cooperative complex formation and targeted ubiquitination. Biophysical characterization revealed distinct linker-dependent cooperativities across the MGPROTAC series, which influenced both cellular permeability and ubiquitination efficiency. Cryo-EM of the most cooperative MGPROTAC uncovered de novo VHL–14-3-3ζ contacts, while molecular dynamics simulations rationalize the stabilizing interactions underlying cooperativity. Strikingly, fine-tuning linker design enables selective ubiquitination of distinct complex subunits. These findings establish a structural and mechanistic framework for integrating molecular glue and PROTAC principles, expanding the scope of drug discovery to previously intractable protein complexes.
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Jul 2026
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[25452, 32707]
Open Access
Abstract: Antimicrobial resistance is driving the search for new antibiotics and a greater understanding of their mechanism of action. Doxycycline is amongst the most-prescribed antimicrobials. It demonstrates a particularly low minimum inhibitory concentration against the zoonotic pathogen Coxiella burnetii. Doxycycline canonically targets the bacterial ribosome by blocking tRNA binding at the decoding centre (A site) of the small subunit. Using cryo-electron microscopy, we analysed doxycycline binding to C. burnetii and Escherichia coli ribosomes. Both structures reveal doxycycline binding at the exit tunnel in the large subunit. In C. burnetii three doxycycline molecules stack to block the tunnel. In E. coli one doxycycline molecule triggers a major change in the conformation of the ribosome. This rearrangement of the peptidyl transferase centre blocks tRNA binding and nascent chain accommodation, abolishing interactions that are fundamental to ribosome function. We identify a distinct ribosomal protein in the C. burnetii large subunit and characterise an additional member of the prokaryotic ribosome hibernation-promoting factor family. These insights into ribosome function and antibiotic action may aid the development of new ribosome inhibitor antibiotics.
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Jun 2026
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Douglas S. M.
Ferreira
,
Katie
Mclennan
,
Calum
Diamond
,
Melanie
Vollmar
,
Wasim
Kiyani
,
D. Sean
Froese
,
Jola
Kopec
,
Henry J.
Bailey
,
Rod
Chalk
,
Arnaud
Basle
,
Jonathan M.
Elkins
,
Jesse
Coker
,
Wyatt W.
Yue
,
Thomas J.
Mccorvie
Diamond Proposal Number(s):
[34172]
Open Access
Abstract: Human methionine synthase (MTR) is an essential enzyme of one carbon metabolism. Consisting of a catalytic N-half and a cobalamin binding C-half, MTR utilises this intricate organometallic cofactor in the methyl transfer from methyltetrahydrofolate to homocysteine producing methionine. Cobalamin loading into MTR, and its subsequent activation, requires methylmalonic aciduria and homocystinuria Type D (MMADHC) protein and methionine synthase reductase (MTRR), respectively. However, the molecular basis of cobalamin binding and activation of human MTR aided by MMADHC and MTRR remains unknown. Here, using cryo-electron microscopy, we determine structures of human MTR in its apo, and cobalamin bound states. Apo MTR adopts a conformation where the two halves of the enzyme act independently with the C-half posed to bind cobalamin. Binding of cobalamin and its activation causes conformational changes in MTR that result in a flexible catalytically active state. AlphaFold predictions, validated by interaction studies, show that MMADHC interacts with the C-half of apo MTR to facilitate cobalamin loading. Unexpectedly we found that MTRR interacts at two distinct sites within the C-half of MTR which may aid in activation. Collectively these findings lay the groundwork to uncover the mechanisms through how MMADHC and MTRR coordinate cobalamin loading and activation of human MTR.
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May 2026
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Krios IV-Titan Krios IV at Diamond
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Rosie M.
Mundy
,
Kasim
Waraich
,
Emily A.
Bates
,
Pierre J.
Rizkallah
,
Alexander T.
Baker
,
Mark T.
Young
,
Edward
Morris
,
Paula C. A.
Da Fonseca
,
Carly M.
Bliss
,
David
Matthews
,
David
Bhella
,
Alan L.
Parker
Diamond Proposal Number(s):
[31827]
Open Access
Abstract: Adenoviruses are widely used as vectors for subunit vaccines and oncolytic therapies. Efficient vectors must infect target cells and deliver therapeutic transgenes at high levels. Species D adenoviruses, such as human adenovirus type 10 (HAdV-D10), are promising candidates due to low seroprevalence in humans. Here, we present the cryo-electron microscopy structure of the HAdV-D10 capsid alongside transcriptomic profiling of infected cells to inform vector development. The fiber shaft, essential for cell entry, was resolved at 10 Å, revealing a previously uncharacterized ‘umbrella’ motif. Viral transcript analysis using an ORF-centric pipeline uncovered key differences from HAdV-C5, including abundant expression of a transcript encoding a protein equivalent to mature protein VII. These findings highlight the importance of detailed vector characterization prior to clinical translation and support the advancement of HAdV-D10 as a next-generation platform for gene delivery and vaccine development.
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Apr 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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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[34312]
Open Access
Abstract: Microtubule nucleation by the γ-tubulin ring complex (γTuRC) is spatiotemporally regulated and in higher eukaryotes is thought to involve a transition from an inactive open to an active closed conformation that matches the microtubule geometry. However, γTuRC activators only promote a partially closed conformation, raising the question of whether complete closure is required for activation. Combining in vitro nucleation assays and cryo-EM, we find that centrosomin motif 1 (CM1), a conserved element of several γTuRC regulators, potently accelerates human γTuRC-mediated microtubule nucleation by facilitating complete closure of γTuRC as the nascent microtubule assembles. A 3.7 Å cryo-EM structure identifies the γTuRC latch and several interactions involved in conformational closure. Notably, the distinct subunits that keep γTuRC open and inactive in higher eukaryotes also participate in its closure and activation. This work provides additional insight into the logic of the human γTuRC architecture and its activation by CM1.
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Mar 2026
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