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Instruments / Technical Area	Publication Details	Published
Krios II-Titan Krios II at Diamond	Probing the biogenesis pathway and dynamics of thylakoid membranes Tuomas Huokko , Tao Ni , Gregory F. Dykes , Deborah M. Simpson , Philip Brownridge , Fabian D. Conradi , Robert J. Beynon , Peter J. Nixon , Conrad W. Mullineaux , Peijun Zhang , Lu-Ning Liu Nature Communications 12 DOI: 10.1038/s41467-021-23680-1 Diamond Proposal Number(s): [21004] Open Access Show Abstract ▼ Abstract: How thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.	Jun 2021
Krios I-Titan Krios I at Diamond Krios II-Titan Krios II at Diamond Krios III-Titan Krios III at Diamond	CryoET structures of immature HIV Gag reveal six-helix bundle Luiza Mendonca , Dapeng Sun , Jiying Ning , Jiwei Liu , Abhay Kotecha , Mateusz Olek , Thomas Frosio , Xiaofeng Fu , Benjamin A. Himes , Alex B. Kleinpeter , Eric O. Freed , Jing Zhou , Christopher Aiken , Peijun Zhang Communications Biology 4 DOI: 10.1038/s42003-021-01999-1 Diamond Proposal Number(s): [18477, 21005, 21004] Open Access Show Abstract ▼ Abstract: Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.	Apr 2021
Krios I-Titan Krios I at Diamond	Alternative architecture of the E. coli chemosensory array Alister Burt , C. Keith Cassidy , Phillip J. Stansfeld , Irina Gutsche Biomolecules 11 DOI: 10.3390/biom11040495 Open Access Show Abstract ▼ Abstract: Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here, we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays appear to exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.	Mar 2021
Krios IV-Titan Krios IV at Diamond	DNA origami signposts for identifying proteins on cell membranes by electron cryotomography Emma Silvester , Benjamin Vollmer , Vojtech Prazak , Daven Vasishtan , Emily A. Machala , Catheryne Whittle , Susan Black , Jonathan Bath , Andrew J. Turberfield , Kay Grunewald , Lindsay A. Baker Cell 184, 1110 - 1121.e16 DOI: 10.1016/j.cell.2021.01.033 Diamond Proposal Number(s): [20223] Open Access Show Abstract ▼ Abstract: Electron cryotomography (cryoET), an electron cryomicroscopy (cryoEM) modality, has changed our understanding of biological function by revealing the native molecular details of membranes, viruses, and cells. However, identification of individual molecules within tomograms from cryoET is challenging because of sample crowding and low signal-to-noise ratios. Here, we present a tagging strategy for cryoET that precisely identifies individual protein complexes in tomograms without relying on metal clusters. Our method makes use of DNA origami to produce “molecular signposts” that target molecules of interest, here via fluorescent fusion proteins, providing a platform generally applicable to biological surfaces. We demonstrate the specificity of signpost origami tags (SPOTs) in vitro as well as their suitability for cryoET of membrane vesicles, enveloped viruses, and the exterior of intact mammalian cells.	Feb 2021
Krios III-Titan Krios III at Diamond	High-light versus low-light: Effects on paired photosystem II supercomplex structural rearrangement in pea plants Alessandro Grinzato , Pascal Albanese , Roberto Marotta , Paolo Swuec , Guido Saracco , Martino Bolognesi , Giuseppe Zanotti , Cristina Pagliano International Journal Of Molecular Sciences 21 DOI: 10.3390/ijms21228643 Diamond Proposal Number(s): [19714] Open Access Show Abstract ▼ Abstract: In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.	Nov 2020
E02-JEM ARM 300CF	Bonding and microstructure evolution in electromagnetic pulse welding of hardenable Al alloys Z. Li , E. Beslin , A.j. Den Bakker , G. Scamans , M. Danaie , C. A. Williams , H. Assadi Journal Of Materials Processing Technology DOI: 10.1016/j.jmatprotec.2020.116965 Diamond Proposal Number(s): [19064] Show Abstract ▼ Abstract: Electromagnetic pulse welding (EMPW) is a promising solid-state joining process, offering fast and strong bonding with no heat affected zone. Despite the growing interest in this process, there is little understanding of the dynamic phenomena that lead to bonding and microstructural changes during EMPW of key engineering materials such as age-hardenable aluminium alloys. This study combines experiments with numerical modelling of plastic deformation to provide an insight to these phenomena in joining of a high-strength aluminium alloy in the T4 and T6 temper conditions. Initially, bonding criteria are postulated in view of the calculated plastic strain at the interface of the T4 sample. These criteria are then used for the prediction of the extent of bonded interfaces for different sets of materials and process parameters. The predictions are shown to be in quantitative agreement with the experimental results for the T6 sample. The corresponding microstructural studies show that bonding is associated with remarkable microstructural changes in the samples, including dissolution of precipitates, formation of high-angle boundaries, and recrystallisation, especially near the bonded interfaces. Moreover, the results of post-weld heat treatments and mechanical testing demonstrate that the impact-induced deformation in EMPW can also influence subsequent precipitations, hence result in improved properties of the entire sample, in a way not achievable by conventional age hardening treatments.	Nov 2020
Krios III-Titan Krios III at Diamond	Assembly intermediates of orthoreovirus captured in the cell Geoff Sutton , Dapeng Sun , Xiaofeng Fu , Abhay Kotecha , Corey W. Hecksel , Daniel K. Clare , Peijun Zhang , David I. Stuart , Mark Boyce Nature Communications 11 DOI: 10.1038/s41467-020-18243-9 Diamond Proposal Number(s): [18477] Open Access Show Abstract ▼ Abstract: Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected ‘single shelled’ intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.	Sep 2020
B24-Cryo Soft X-ray Tomography	CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging Michael A. Phillips , Maria Harkiolaki , David Miguel Susano Pinto , Richard M. Parton , Ana Palanca , Manuel Garcia-Moreno , Ilias Kounatidis , John W. Sedat , David I. Stuart , Alfredo Castello , Martin J. Booth , Ilan Davis , Ian Dobbie Optica 7 DOI: 10.1364/OPTICA.393203 Diamond Proposal Number(s): [18319, 18757] Open Access Show Abstract ▼ Abstract: Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.	Jul 2020

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