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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I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[19832]
Open Access
Abstract: We review the structural and functional characteristics of bacteriophages and bacteriocins (diffocins) that specifically target Clostridioides difficile, a significant healthcare concern due to its role in nosocomial infections. The advent of modern cryogenic electron microscopy (cryoEM) has revolutionized our understanding of these contractile injection systems, providing high-resolution insights into their mechanisms. We compare the structures of C. difficile phages and diffocins, highlighting their adaptations for penetrating the Gram-positive bacterial cell envelope – including the cell membrane, cell wall and proteinaceous surface layer. Diffocins, simpler in structure, utilize a combination of mechanical and enzymatic strategies, while some phages like ΦCD508 may rely on mechanical force alone. This review delves into the assembly and function of key components such as the contractile sheath, baseplate and receptor-binding proteins, offering a framework for engineering precision antimicrobials. We also present new experimental results, including refined cryoEM structures of the ΦCD508 pre- and post-contracted tail, a novel spontaneously contracted conformation and an X-ray crystal structure of a phage receptor-binding protein domain. This work underscores the potential of cryoEM in advancing our understanding of phage biology and its applications in developing targeted therapies against C. difficile.
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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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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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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Bacteroides thetaiotaomicron (B. theta) is a model Bacteroidota of the healthy human gut microbiota and a specialist in glycan utilisation. Like other Bacteroides, B. theta has many highly regulated polysaccharide utilisation loci (PUL) that encode outer membrane (OM) TonB-dependent transporters (SusC), closely associated “lid” lipoproteins (SusD), and additional surface-exposed lipoproteins (SLPs) that bind and partially degrade specific glycans derived from host cells, diet, or other microbiota members. The canonical starch PUL products are thought to form a dynamic complex in the presence of starch. However, other PULs form stable complexes in the absence of substrate (recently named “utilisomes”), with additional surface lipoproteins tightly associated with the core SusCD complex. In this study, we characterised the B. theta dextran utilisome, with a SusCDdex core and an associated glycoside hydrolase (GHdex) and surface glycan binding protein (SBGPdex). Via X-ray crystallography we solved high-resolution structures of SBGPdex in isolation and SusDdex and GHdex bound to dextran oligosaccharides. We used isothermal titration calorimetry (ITC) to quantify ligand binding of wild type and mutant SLPs. We further used single particle cryo-EM of the catalytically inactive dextran utilisome to visualise open and closed states of the complex. Three occupied dextran binding sites were observed across SusCdex, SusDdex and GHdex, with substrate observed in both open and closed states of SusDdex. 3D variability analysis showed a minority of particles in the process of SusDdex lid closure. Together our work defines commonalities and differences across utilisomes dedicated to the import of simple glycans.
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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 I-Titan Krios I at Diamond
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Ane
Martinez-Castillo
,
Andrea
Aebischer
,
Philippine
Toneatti
,
Lifei
Fu
,
Damien
Vitour
,
Corinne
Sailleau
,
Bernd
Hoffmann
,
Kati
Franzke
,
Michael
Eschbaumer
,
Saskia
Weber
,
Eva
Calvo Pinilla
,
Javier
Ortego
,
David
Gil-Carton
,
Emmanuel
Bréard
,
Stéphan
Zientara
,
Jeroen
Kortekaas
,
Martin
Beer
,
Nicola G. A.
Abrescia
Diamond Proposal Number(s):
[31586]
Open Access
Abstract: African horse sickness virus (AHSV) is a lethal equine pathogen with no licensed vaccine other than vaccines containing attenuated virus, which pose safety risks. Endemic to sub-Saharan Africa, AHSV has caused epizootics in Spain and Portugal, Cyprus, Morocco, the Middle East, India and Pakistan and, most recently, Thailand. Here, we resolve the 3.11 Å cryo-EM structure of full-length VP2 from AHSV serotype 4, adopting its native triskelion architecture and shedding light on an α-helical domain anchoring the triskelion core, which is absent in other structurally characterized orbiviruses. Structure-guided mapping identified a subdomain of VP2 as a key target of neutralizing antibodies. Displayed on nanoparticles using the SpyCatcher/SpyTag technology, the domain conferred complete protection from clinical disease after viral challenge infection in mice and elicited robust and long-lasting immune responses in horses, the target species of AHSV. These findings provide a structural blueprint for the next generation of recombinant vaccines against AHSV and related orbiviruses.
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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):
[37221]
Open Access
Abstract: Biocatalytic cascades offer a promising route for CO2-fixation into valuable chemicals, addressing the urgent need for efficient, sustainable technologies to reduce CO2 emissions. This paper describes an enzymatic route converting gaseous CO2 and acetaldehyde into enantiopure lactic acid, widely used in diverse industries. A newly characterized pyruvate decarboxylase from Neoasia chiangmaiensis (NcPDC) enabled acetaldehyde carboxylation to pyruvate. To suppress the competing carboligation to acetoin, acetaldehyde was reversibly trapped with Tris. Pyruvate was reduced to lactate by lactate dehydrogenase, coupled with glucose dehydrogenase for NADH regeneration via D-glucose oxidation to D-gluconic acid. Up to 65% lactate yield was achieved. Repeated acetaldehyde dosing resulted in a 27 mM titer, representing a >100-fold improvement over previous reports. At 0.5 L scale, using a gas mixture mimicking industrial-grade CO2, we obtained 21 mM D-(–)-lactic acid, 42% yield and >98% e.e., demonstrating scalability and robustness. Finally, replacing the D-(–)-selective lactate dehydrogenase with an L-(+)-selective variant at small scale enabled production of L-(+)-lactic acid at 41% yield and >93% e.e, allowing switchable access to either enantiomer. A volumetric productivity of 1.1 × 10−2 g L−1 h−1 ranks among the most efficient minimal enzymatic routes developed to date for CO2-to-lactate conversion.
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Jun 2026
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Krios III-Titan Krios III at Diamond
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Ho Fong
Leong
,
Giovanni
Consoli
,
Geoffry A.
Davis
,
Ben
Hancox-Lachman
,
Kenta
Renard
,
Fiazall
Tufail
,
Lauren E.
Lee
,
Lucas
Gautier
,
James W.
Murray
,
Andrea
Fantuzzi
,
A. William
Rutherford
Diamond Proposal Number(s):
[33230]
Open Access
Abstract: Far-red light photoacclimation enables some cyanobacteria to survive in white-light-depleted environments by extending the red limit of photosynthesis. In far-red Photosystem II, paralogous subunits replace their canonical counterparts, allowing the incorporation of some chlorophyll f molecules and one chlorophyll d that are red-shifted and spectrally distinct from the chlorophyll a manifold, and from each other. Here, we present a comparative study of far-red Photosystem II from Chroococcidiopsis thermalis PCC 7203 and Calothrix sp. NIES-3974. In C. thermalis, the cryo-electron microscopy structure reveals the far-red-exclusive subunit, PsbH2’, which forms part of a chlorophyll f binding site. We also assign four chlorophyll f sites using sequence comparisons and electrostatic potential analyses. In Calothrix, psbH2’ is absent, and the same analyses show that only two of these chlorophyll f sites are present. Comparative phylogenetic, structural, and spectroscopic analyses allow the assignment of specific wavelengths to all the red-shifted chlorophylls. This provides the framework needed to model excitation energy transfer in far-red Photosystem II, and to understand the conserved features that allow survival under far-red light.
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Jun 2026
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