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34 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4 [Next/Last]

Instruments / Technical Area	Publication Details	Published
Krios II-Titan Krios II at Diamond Krios III-Titan Krios III at Diamond	Unveiling the structural spectrum of SARS-CoV-2 fusion by in situ cryo-ET Caner Akil , Jialu Xu , Juan Shen , Peijun Zhang Nature Communications 16 DOI: 10.1038/s41467-025-60406-z Diamond Proposal Number(s): [29812] Open Access Show Abstract ▼ 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.	Jun 2025
Krios II-Titan Krios II at Diamond	Rubisco packaging and stoichiometric composition of the native β-carboxysome in Synechococcus elongatus PCC7942 Yaqi Sun , Yuewen Sheng , Tao Ni , Xingwu Ge , Joscelyn Sarsby , Philip J. Brownridge , Kang Li , Nathan Hardenbrook , Gregory F. Dykes , Nichola Rockliffe , Claire E. Eyers , Peijun Zhang , Lu-Ning Liu Plant Physiology DOI: 10.1093/plphys/kiae665 Diamond Proposal Number(s): [21004] Open Access Show Abstract ▼ Abstract: Carboxysomes are anabolic bacterial microcompartments that play an essential role in CO2 fixation in cyanobacteria. This self-assembling proteinaceous organelle uses a polyhedral shell constructed by hundreds of shell protein paralogs to encapsulate the key CO2-fixing enzymes Rubisco and carbonic anhydrase. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding carboxysome formation and overall functionality. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also detected Rubisco binding with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using Quantification conCATamer (QconCAT)-based quantitative mass spectrometry, we determined the absolute stoichiometric composition of the entire β-carboxysome. This study provides insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.	Dec 2024
Krios III-Titan Krios III at Diamond	CryoET reveals actin filaments within platelet microtubules Chisato Tsuji , Marston Bradshaw , Megan F. Allen , Molly L. Jackson , Judith Mantell , Ufuk Borucu , Alastair W. Poole , Paul Verkade , Ingeborg Hers , Danielle M. Paul , Mark P. Dodding Nature Communications 15 DOI: 10.1038/s41467-024-50424-8 Diamond Proposal Number(s): [25452, 32707] Open Access Show Abstract ▼ Abstract: Crosstalk between the actin and microtubule cytoskeletons is important for many cellular processes. Recent studies have shown that microtubules and F-actin can assemble to form a composite structure where F-actin occupies the microtubule lumen. Whether these cytoskeletal hybrids exist in physiological settings and how they are formed is unclear. Here, we show that the short-crossover Class I actin filament previously identified inside microtubules in human HAP1 cells is cofilin-bound F-actin. Lumenal F-actin can be reconstituted in vitro, but cofilin is not essential. Moreover, actin filaments with both cofilin-bound and canonical morphologies reside within human platelet microtubules under physiological conditions. We propose that stress placed upon the microtubule network during motor-driven microtubule looping and sliding may facilitate the incorporation of actin into microtubules.	Jul 2024
Talos-Talos Arctica at Diamond	Cryo-tomography and 3D electron diffraction reveal the polar habit and chiral structure of the malaria pigment crystal hemozoin Paul B. Klar , David G. Waterman , Tim Gruene , Debakshi Mullick , Yun Song , James B. Gilchrist , C. David Owen , Wen Wen , Idan Biran , Lothar Houben , Neta Regev-Rudzki , Ron Dzikowski , Noa Marom , Lukas Palatinus , Peijun Zhang , Leslie Leiserowitz , Michael Elbaum Acs Central Science DOI: 10.1021/acscentsci.4c00162 Diamond Proposal Number(s): [21004, 29812] Open Access Show Abstract ▼ Abstract: Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. The crystalline structure of hemozoin was established by X-ray powder diffraction using a synthetic analog, β-hematin. Here, we apply emerging methods of in situ cryo-electron tomography and 3D electron diffraction to obtain a definitive structure of hemozoin directly from ruptured parasite cells. Biogenic hemozoin crystals take a striking polar morphology. Like β-hematin, the unit cell contains a heme dimer, which may form four distinct stereoisomers: two centrosymmetric and two chiral enantiomers. Diffraction analysis, supported by density functional theory analysis, reveals a selective mixture in the hemozoin lattice of one centrosymmetric and one chiral dimer. Absolute configuration has been determined by morphological analysis and confirmed by a novel method of exit-wave reconstruction from a focal series. Atomic disorder appears on specific facets asymmetrically, and the polar morphology can be understood in light of water binding. Structural modeling of the heme detoxification protein suggests a function as a chiral agent to bias the dimer formation in favor of rapid growth of a single crystalline phase. The refined structure of hemozoin should serve as a guide to new drug development.	Jul 2024
Krios III-Titan Krios III at Diamond Krios IV-Titan Krios IV at Diamond	A closer look at how cells package DNA Diamond Light Source Diamond Proposal Number(s): [29812] Show Abstract ▼ Abstract: Our cells use an ensemble of histone proteins to fold and package the DNA genome into the nucleus. Histones also determine whether to expose DNA to enzymes to allow processes like gene expression, replication, and repair to occur. Although many in vitro studies have explored the mechanism histones use to fold and package DNA into higher-order structures called chromatin, less is known about chromatin organisation inside the nucleus of intact cells, and understanding this phenomenon could be key to understanding multiple DNA-associated processes. Recent advances in cryo-electron tomography have enabled scientists to observe these structures within the nucleus of rapidly cryopreserved cells. Reporting in Nature Communications, scientists at the University of Oxford collaborated with the electron Bio-Imaging Centre (eBIC) at the Diamond Light Source to capture chromatin in the nucleus of immune T cells, revealing that DNA is folded into more flexible and heterogenous fibres than previously modelled. Their experiments lay the groundwork for future studies into the roles of chromatin in health and disease.	Dec 2023
Krios III-Titan Krios III at Diamond Krios IV-Titan Krios IV at Diamond	Structure of native chromatin fibres revealed by Cryo-ET in situ Zhen Hou , Frank Nightingale , Yanan Zhu , Craig Macgregor-Chatwin , Peijun Zhang Nature Communications 14 DOI: 10.1038/s41467-023-42072-1 Diamond Proposal Number(s): [29812] Open Access Show Abstract ▼ Abstract: The structure of chromatin plays pivotal roles in regulating gene transcription, DNA replication and repair, and chromosome segregation. This structure, however, remains elusive. Here, using cryo-FIB and cryo-ET, we delineate the 3D architecture of native chromatin fibres in intact interphase human T-lymphoblasts and determine the in situ structures of nucleosomes in different conformations. These chromatin fibres are not structured as uniform 30 nm one-start or two-start filaments but are composed of relaxed, variable zigzag organizations of nucleosomes connected by straight linker DNA. Nucleosomes with little H1 and linker DNA density are distributed randomly without any spatial preference. This work will inspire future high-resolution investigations on native chromatin structures in situ at both a single-nucleosome level and a population level under many different cellular conditions in health and disease.	Oct 2023
Krios III-Titan Krios III at Diamond	An improved method for growing primary neurons on electron microscopy grids co-cultured with astrocytes Ishika Kumar , Anju Paudyal , Anna Kádková , Michelle Stewart , Jakob Balslev Sørensen , Julika Radecke International Journal Of Molecular Sciences 24 DOI: 10.3390/ijms242015191 Diamond Proposal Number(s): [29869] Open Access Show Abstract ▼ Abstract: With the increasing popularity of cryo-electron tomography (cryo-ET) in recent years, the quest to establish a method for growing primary neurons directly on electron microscopy grids (EM grids) has been ongoing. Here we describe a straightforward way to establish a mature neuronal network on EM grids, which includes formation of synaptic contacts. These synapses were thin enough to allow for direct visualization of small filaments such as SNARE proteins tethering the synaptic vesicle (SV) to the active zone plasma membrane on a Titan Krios without prior focused ion-beam milling.	Oct 2023
Krios I-Titan Krios I at Diamond Krios III-Titan Krios III at Diamond Krios IV-Titan Krios IV at Diamond	ChAdOx1 COVID vaccines express RBD open prefusion SARS-CoV-2 spikes on the cell surface Tao Ni , Luiza Mendonca , Yanan Zhu , Andrew Howe , Julika Radecke , Pranav M. Shah , Yuewen Sheng , Anna-Sophia Krebs , Helen M. E. Duyvesteyn , Elizabeth Allen , Teresa Lambe , Cameron Bisset , Alexandra Spencer , Susan Morris , David I. Stuart , Sarah Gilbert , Peijun Zhang Iscience DOI: 10.1016/j.isci.2023.107882 Diamond Proposal Number(s): [26987] Open Access Show Abstract ▼ Abstract: Vaccines against SARS-CoV-2 have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1 vectored HexaPro stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild-type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.	Sep 2023
Krios II-Titan Krios II at Diamond Krios III-Titan Krios III at Diamond	Structure of the native chemotaxis core signaling unit from phage E-protein lysed E. coli cells C. Keith Cassidy , Zhuan Qin , Thomas Frosio , Khoosheh Gosink , Zhengyi Yang , Mark S. P. Sansom , Phillip J. Stansfeld , John S. Parkinson , Peijun Zhang Mbio DOI: 10.1128/mbio.00793-23 Diamond Proposal Number(s): [21004, 22941, 22910] Open Access Show Abstract ▼ Abstract: Motile bacteria employ conserved chemotaxis networks to detect chemical gradients in their surroundings and effectively regulate their locomotion, enabling the location of essential nutrients and other important biological niches. The sensory apparatus of the chemotaxis pathway is an array of core-signaling units (CSUs) composed of transmembrane chemoreceptors, the histidine kinase CheA and an adaptor protein, CheW. Although chemotaxis pathways represent the best understood signaling systems, a detailed mechanistic understanding of signal transduction has been hindered by the lack of a complete structural picture of the CSU and extended array. In this study, we present the structure of the complete CSU from phage φX174 E protein lysed Escherichia coli cells, determined using cryo-electron tomography and sub-tomogram averaging to 12-Å resolution. Using AlphaFold2, we further predict the atomic structures of the CSU’s constituent proteins as well as key protein-protein interfaces, enabling the assembly an all-atom CSU model, which we conformationally refine using our cryo-electron tomography map. Molecular dynamics simulations of the resulting model provide new insight into the periplasmic organization of the complex, including novel interactions between neighboring receptor ligand-binding domains. Our results further elucidate previously unresolved interactions between individual CheA domains, including an anti-parallel P1 dimer and non-productive binding mode between P1 and P4, enhancing our understanding of the structural mechanisms underlying CheA signaling and regulation.	Sep 2023
Krios I-Titan Krios I at Diamond Krios IV-Titan Krios IV at Diamond	Neutral sphingomyelinase 2 is required for HIV-1 maturation Abdul A. Waheed , Yanan Zhu , Eva Agostino , Lwar Naing , Yuta Hikichi , Ferri Soheilian , Seung-Wan Yoo , Yun Song , Peijun Zhang , Barbara S. Slusher , Norman J. Haughey , Eric O. Freed Proceedings Of The National Academy Of Sciences 120 DOI: 10.1073/pnas.2219475120 Diamond Proposal Number(s): [29812] Show Abstract ▼ Abstract: HIV-1 assembly occurs at the inner leaflet of the plasma membrane (PM) in highly ordered membrane microdomains. The size and stability of membrane microdomains is regulated by activity of the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) that is localized primarily to the inner leaflet of the PM. In this study, we demonstrate that pharmacological inhibition or depletion of nSMase2 in HIV-1-producer cells results in a block in the processing of the major viral structural polyprotein Gag and the production of morphologically aberrant, immature HIV-1 particles with severely impaired infectivity. We find that disruption of nSMase2 also severely inhibits the maturation and infectivity of other primate lentiviruses HIV-2 and simian immunodeficiency virus, has a modest or no effect on nonprimate lentiviruses equine infectious anemia virus and feline immunodeficiency virus, and has no effect on the gammaretrovirus murine leukemia virus. These studies demonstrate a key role for nSMase2 in HIV-1 particle morphogenesis and maturation.	Jul 2023

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