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

Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond	Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae James M. Parkhurst , Adam D. Crawshaw , C. Alistair Siebert , Maud Dumoux , C. David Owen , Pedro Nunes , David Waterman , Thomas Glen , David I. Stuart , James H. Naismith , Gwyndaf Evans Iucrj 10 DOI: 10.1107/S2052252523001902 Open Access Show Abstract ▼ Abstract: Three-dimensional electron diffraction (3DED) from nanocrystals of biological macromolecules requires the use of very small crystals. These are typically less than 300 nm-thick in the direction of the electron beam due to the strong interaction between electrons and matter. In recent years, focused-ion-beam (FIB) milling has been used in the preparation of thin samples for 3DED. These instruments typically use a gallium liquid metal ion source. Inductively coupled plasma (ICP) sources in principle offer faster milling rates. Little work has been done to quantify the damage these sources cause to delicate biological samples at cryogenic temperatures. Here, an analysis of the effect that milling with plasma FIB (pFIB) instrumentation has on lysozyme crystals is presented. This work evaluates both argon and xenon plasmas and compares them with crystals milled with a gallium source. A milling protocol was employed that utilizes an overtilt to produce wedge-shaped lamellae with a shallow thickness gradient which yielded very thin crystalline samples. 3DED data were then acquired and standard data-processing statistics were employed to assess the quality of the diffraction data. An upper bound to the depth of the pFIB-milling damage layer of between 42.5 and 50 nm is reported, corresponding to half the thickness of the thinnest lamellae that resulted in usable diffraction data. A lower bound of between 32.5 and 40 nm is also reported, based on a literature survey of the minimum amount of diffracting material required for 3DED.	May 2023
Krios I-Titan Krios I at Diamond	Cryo-electron tomography of intact cardiac muscle reveals myosin binding protein-C linking myosin and actin filaments Xinrui Huang , Iratxe Torre , Michele Chiappi , Zhan Yin , Anupama Vydyanath , Shuangyi Cao , Oliver Raschdorf , Morgan Beeby , Bonnie Quigley , Pieter P. De Tombe , Jun Liu , Edward P. Morris , Pradeep K. Luther Journal Of Muscle Research And Cell Motility 189 DOI: 10.1007/s10974-023-09647-3 Diamond Proposal Number(s): [18092] Open Access Show Abstract ▼ Abstract: Myosin binding protein C (MyBP-C) is an accessory protein of the thick filament in vertebrate cardiac muscle arranged over 9 stripes of intervals of 430 Å in each half of the A-band in the region called the C-zone. Mutations in cardiac MyBP-C are a leading cause of hypertrophic cardiomyopathy the mechanism of which is unknown. It is a rod-shaped protein composed of 10 or 11 immunoglobulin- or fibronectin-like domains labelled C0 to C10 which binds to the thick filament via its C-terminal region. MyBP-C regulates contraction in a phosphorylation dependent fashion that may be through binding of its N-terminal domains with myosin or actin. Understanding the 3D organisation of MyBP-C in the sarcomere environment may provide new light on its function. We report here the fine structure of MyBP-C in relaxed rat cardiac muscle by cryo-electron tomography and subtomogram averaging of refrozen Tokuyasu cryosections. We find that on average MyBP-C connects via its distal end to actin across a disc perpendicular to the thick filament. The path of MyBP-C suggests that the central domains may interact with myosin heads. Surprisingly MyBP-C at Stripe 4 is different; it has weaker density than the other stripes which could result from a mainly axial or wavy path. Given that the same feature at Stripe 4 can also be found in several mammalian cardiac muscles and in some skeletal muscles, our finding may have broader implication and significance. In the D-zone, we show the first demonstration of myosin crowns arranged on a uniform 143 Å repeat.	Apr 2023
Krios III-Titan Krios III at Diamond	Characterization of the rotavirus assembly pathway in situ using cryoelectron tomography Pranav N. M. Shah , James B. Gilchrist , Björn O. Forsberg , Alister Burt , Andrew Howe , Shyamal Mosalaganti , William Wan , Julika Radecke , Yuriy Chaban , Geoff Sutton , David I. Stuart , Mark Boyce Cell Host & Microbe 190 DOI: 10.1016/j.chom.2023.03.004 Diamond Proposal Number(s): [21004] Open Access Show Abstract ▼ Abstract: Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.	Mar 2023
Krios I-Titan Krios I at Diamond Krios II-Titan Krios II at Diamond	Structure and activity of particulate methane monooxygenase arrays in methanotrophs Yanan Zhu , Christopher W. Koo , C. Keith Cassidy , Matthew C. Spink , Tao Ni , Laura C. Zanetti-Domingues , Benji Bateman , Marisa Martin-Fernandez , Juan Shen , Yuewen Sheng , Yun Song , Zhengyi Yang , Amy C. Rosenzweig , Peijun Zhang Nature Communications 13 DOI: 10.1038/s41467-022-32752-9 Diamond Proposal Number(s): [21004, 29812] Open Access Show Abstract ▼ Abstract: Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.	Sep 2022
Krios I-Titan Krios I at Diamond	Cryo-electron tomography remote data collection and subtomogram averaging Yuewen Sheng , Kyle Morris , Julika Radecke , Peijun Zhang Journal Of Visualized Experiments DOI: 10.3791/63923 Open Access Show Abstract ▼ Abstract: Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.	Jul 2022
B24-Cryo Soft X-ray Tomography I13-2-Diamond Manchester Imaging Krios I-Titan Krios I at Diamond	SuRVoS 2: accelerating annotation and segmentation for large volumetric bioimage workflows across modalities and scales Avery Pennington , Oliver N. F. King , Win Min Tun , Elaine M. L. Ho , Imanol Luengo , Michele C. Darrow , Mark Basham Frontiers In Cell And Developmental Biology 10 DOI: 10.3389/fcell.2022.842342 Open Access Show Abstract ▼ Abstract: As sample preparation and imaging techniques have expanded and improved to include a variety of options for larger sized and numbers of samples, the bottleneck in volumetric imaging is now data analysis. Annotation and segmentation are both common, yet difficult, data analysis tasks which are required to bring meaning to the volumetric data. The SuRVoS application has been updated and redesigned to provide access to both manual and machine learning-based segmentation and annotation techniques, including support for crowd sourced data. Combining adjacent, similar voxels (supervoxels) provides a mechanism for speeding up segmentation both in the painting of annotation and by training a segmentation model on a small amount of annotation. The support for layers allows multiple datasets to be viewed and annotated together which, for example, enables the use of correlative data (e.g. crowd-sourced annotations or secondary imaging techniques) to guide segmentation. The ability to work with larger data on high-performance servers with GPUs has been added through a client-server architecture and the Pytorch-based image processing and segmentation server is flexible and extensible, and allows the implementation of deep learning-based segmentation modules. The client side has been built around Napari allowing integration of SuRVoS into an ecosystem for open-source image analysis while the server side has been built with cloud computing and extensibility through plugins in mind. Together these improvements to SuRVoS provide a platform for accelerating the annotation and segmentation of volumetric and correlative imaging data across modalities and scales.	Apr 2022
Krios I-Titan Krios I at Diamond	A cryo-ET survey of microtubules and intracellular compartments in mammalian axons Helen E. Foster , Camilla Ventura Santos , Andrew P. Carter Journal Of Cell Biology 221 DOI: 10.1083/jcb.202103154 Diamond Proposal Number(s): [23268, 17434] Open Access Show Abstract ▼ Abstract: The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the internal components of mammalian sensory axons. We determined the polarity of the axonal microtubules (MTs) by combining subtomogram classification and visual inspection, finding MT plus and minus ends are structurally similar. Subtomogram averaging of globular densities in the MT lumen suggests they have a defined structure, which is surprising given they likely contain the disordered protein MAP6. We found the endoplasmic reticulum in axons is tethered to MTs through multiple short linkers. We surveyed membrane-bound cargos and describe unexpected internal features such as granules and broken membranes. In addition, we detected proteinaceous compartments, including numerous virus-like capsid particles. Our observations outline novel features of axonal cargos and MTs, providing a platform for identification of their constituents.	Feb 2022
Krios I-Titan Krios I at Diamond	The pore conformation of lymphocyte perforin Marina E. Ivanova , Natalya Lukoyanova , Sony Malhotra , Maya Topf , Joseph A. Trapani , Ilia Voskoboinik , Helen R. Saibil Science Advances 8 DOI: 10.1126/sciadv.abk3147 Diamond Proposal Number(s): [14704] Open Access Show Abstract ▼ Abstract: Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with proapoptotic granzymes, to bind to a target cell membrane where it oligomerizes and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here, we present a 4-Å resolution cryo–electron microscopy structure of the perforin pore, revealing previously unidentified inter- and intramolecular interactions stabilizing the assembly. During pore formation, the helix-turn-helix motif moves away from the bend in the central beta-sheet to form an intermolecular contact. Cryo–electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerization and membrane insertion, and explain how several pathogenic mutations affect function.	Feb 2022
Krios II-Titan Krios II at Diamond	High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity Tao Ni , Thomas Frosio , Luiza Mendonca , Yuewen Sheng , Daniel Clare , Benjamin A. Himes , Peijun Zhang Nature Protocols 58 DOI: 10.1038/s41596-021-00648-5 Diamond Proposal Number(s): [26464] Show Abstract ▼ Abstract: Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.	Jan 2022
Krios I-Titan Krios I at Diamond Krios II-Titan Krios II at Diamond Krios IV-Titan Krios IV at Diamond	Structure of native HIV-1 cores and their interactions with IP6 and CypA Tao Ni , Yanan Zhu , Zhengyi Yang , Chaoyi Xu , Yuriy Chaban , Tanya Nesterova , Jiying Ning , Till Böcking , Michael W. Parker , Christina Monnie , Jinwoo Ahn , Juan R. Perilla , Peijun Zhang Science Advances 7 DOI: 10.1126/sciadv.abj5715 Diamond Proposal Number(s): [21004, 20223] Open Access Show Abstract ▼ Abstract: The viral capsid plays essential roles in HIV replication and is a major platform engaging host factors. To overcome challenges in study native capsid structure, we used the perfringolysin O to perforate the membrane of HIV-1 particles, thus allowing host proteins and small molecules to access the native capsid while improving cryo–electron microscopy image quality. Using cryo–electron tomography and subtomogram averaging, we determined the structures of native capsomers in the presence and absence of inositol hexakisphosphate (IP6) and cyclophilin A and constructed an all-atom model of a complete HIV-1 capsid. Our structures reveal two IP6 binding sites and modes of cyclophilin A interactions. Free energy calculations substantiate the two binding sites at R18 and K25 and further show a prohibitive energy barrier for IP6 to pass through the pentamer. Our results demonstrate that perfringolysin O perforation is a valuable tool for structural analyses of enveloped virus capsids and interactions with host cell factors.	Nov 2021

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