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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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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E01-JEM ARM 200CF
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Diamond Proposal Number(s):
[45820]
Abstract: Atomic-scale characterisation of thin buried layers in oxide heterostructures is often limited by the reduced sensitivity of conventional Z-contrast high-angle annular dark-field (HAADF) imaging to light elements and low-density regions. Here, a Sc2O3/Er2O3/Si heterostructure was investigated using aberration-corrected scanning transmission electron microscope (STEM) with simultaneous HAADF and integrated differential phase contrast (iDPC) imaging, with site-specific cross-sectional specimens prepared by scanning electron microscope-plasma focused ion beam (SEM-PFIB) using a Xe-ion beam under identical acquisition conditions to enable direct contrast comparison. Although HAADF clearly resolves the heavy rare-earth oxide layers and reveals a broad interfacial region with contrast variations indicative of structural complexity, simultaneous STEM-iDPC imaging resolves a distinct ∼3 nm buried interfacial layer with substantially greater clarity. Additional high-resolution transmission electron microscopy (HRTEM) and electron energy-loss spectroscopy (EELS) analyses reveal oxygen enrichment and local structural ordering within this region, indicating that it is chemically and structurally distinct from both crystalline Si and bulk Er2O3. The combined observations are consistent with an oxygen-rich Er–Si–O transition layer. These results demonstrate the capability of STEM-iDPC to reveal buried interfacial structure and highlight the value of combining HAADF, iDPC, HRTEM and spectroscopy for comprehensive characterisation of oxide heterostructures.
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Jul 2026
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E02-JEM ARM 300CF
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Leyuan
Zhang
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Dongfang
Cheng
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Pu
Zhang
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David G.
Hopkinson
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Zhaozong
Wang
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Ao
Zhang
,
Chen
Li
,
Ran
Wang
,
Rongli
Liu
,
Christopher S.
Allen
,
Johanna
Nelson Weker
,
Yu
Huang
,
Philippe
Sautet
,
Xiangfeng
Duan
Diamond Proposal Number(s):
[32750]
Open Access
Abstract: Lithium–sulfur batteries are fundamentally constrained by the sluggish 16-electron sulfur reduction reaction. Electrocatalytic sulfur reduction reaction is inherently complex, involving multiple lithium polysulfide intermediates (Li2Sn, n = 2–8), each with distinct adsorption and activation requirements, leading to unbalanced polysulfide conversion and severe shuttle effect. Although cascade catalysis has been proposed as a potential solution, the precise pathway and its mechanistic role in regulating polysulfide conversion remain elusive. Here we elucidate and experimentally validate the complete cascade pathway of sulfur reduction on Fe,N,S-codoped holey graphene as a model catalyst. Density functional theory reveals that Fe sites preferentially bind and activate long-chain polysulfides, while N,S-C sites accelerate the conversion of Li2S4 to Li2S2/Li2S. Such site-specific synergy balances sulfur reduction kinetics and suppresses polysulfide accumulation. Combined kinetic analysis and operando Raman spectroscopy directly reveal how synergistic cascade catalysis governs the reaction pathway, modulates key intermediates, and enables balanced polysulfide conversion. Together, these results establish cascade catalysis as a mechanism-driven design strategy for lithium–sulfur battery electrodes, where regulation of the reaction pathway suppresses polysulfide shuttling and enables enhanced cycling stability.
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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
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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
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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E01-JEM ARM 200CF
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Emerson C.
Kohlrausch
,
Christopher
Leist
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Gazi N.
Aliev
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Mohsen
Danaie
,
Matthew
Young
,
Madasamy
Thangamuthu
,
Yifan
Chen
,
William J.
Cull
,
Wolfgang
Theis
,
Ute
Kaiser
,
Andrei N.
Khlobystov
,
Jesum
Alves Fernandes
Diamond Proposal Number(s):
[37379, 38763]
Abstract: Understanding how catalytically active sites emerge and evolve under working conditions is a fundamental challenge that limits the rational design of heterogeneous catalysts. Here, we directly visualize the transformation between alloyed PtNi and phase-separated Pt-NiO nanoclusters during hydrogen evolution. Using in situ low-voltage aberration-corrected electron microscopy, with the electron beam serving as both the stimulus and probe, we track the formation of active sites under low-water-vapor conditions. PtNi nanoclusters were assembled with controlled mixing of the atoms, resulting in two distinct configurational entropy states. Under reaction conditions, the transformation of bimetallic nanoclusters shifts from an entropically stabilized alloy to an enthalpically favored phase-separated configuration, controlled by oxygen availability and by a critical nucleus size. The atomic dynamics observed in real space correlate directly with catalytic performance, where the low-entropy Pt-NiO state achieves a record hydrogen evolution mass activity of 11.1 A/mgPt due to a high density of interfacial sites that promote water dissociation on NiO and efficient hydrogen adsorption on Pt atoms.
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Jun 2026
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E03-EM03E ePSIC Microscope (JEOL FIB)
I13-1-Coherence
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Diamond Proposal Number(s):
[42138, 39271]
Abstract: Characterizing the 3D morphology of nano- and micro-scale precipitates in metallic materials remains challenging. By combining 3D focused ion beam imaging with nondestructive synchrotron x-ray ptychographic tomography, this study pioneers a multi-modal imaging approach that unveils the 3D morphology of NiTi2 precipitates and a previously unknown cross-linked network of Ni4Ti3 precipitates in the nickel–titanium alloys, achieving a spatial resolution of 52 nm. Key discoveries challenge long-standing assumptions: Ni4Ti3 precipitates can form a network rather than isolated ellipsoids through three distinct cross-linking modes. Their shapes are not perfectly lenticular due to overlapping stress fields and loss of coherency. The 3D morphology of NiTi2 precipitates shows that they are primarily spherical and governed by interfacial energy minimization. The competitive growth mechanisms are captured via phase field simulation. These insights deepen the understanding of precipitate growth in NiTi alloys and establish a new paradigm for 3D microstructural imaging.
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Jun 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 I-Titan Krios I at Diamond
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Carys
Williams
,
Laura M.
Nocka
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George
Hedger
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Pragya
Parashara
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Els
Pardon
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Naomi R.
Latorraca
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Ganesh V.
Pusapati
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Parijat
Sarkar
,
Dorothy
Lartey
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Lei
Gao
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Ljiljana
Milenkovic
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Rod
Chalk
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Jan
Steyaert
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Susan
Marqusee
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Loic
Carrique
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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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