B24-Cryo Soft X-ray Tomography
I13-2-Diamond Manchester Imaging
Krios I-Titan Krios I at Diamond
|
Open Access
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 II-Titan Krios II at Diamond
|
Diamond Proposal Number(s):
[26464]
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
|
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
Diamond Proposal Number(s):
[21004, 20223]
Open Access
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
|
|
Scios-Scios at Diamond
|
Open Access
Abstract: Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are increasingly used for macromolecular structure determination in situ. Here, we introduce a set of computational tools and resources designed to enable flexible approaches to STA through increased automation and simplified metadata handling. We create a bidirectional interface between the Dynamo software package and the Warp-Relion-M pipeline, providing a framework for ab initio and geometrical approaches to multiparticle refinement in M. We illustrate the power of working within this framework by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles (VLPs), and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we provide a comprehensive, step-by-step guide to obtaining a 3.4-Å reconstruction from EMPIAR-10164. The guide is hosted on https://teamtomo.org/, a collaborative online platform we establish for sharing knowledge about cryo-ET.
|
Aug 2021
|
|
Krios II-Titan Krios II at Diamond
|
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
Diamond Proposal Number(s):
[21004]
Open Access
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
|
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
Diamond Proposal Number(s):
[18477, 21005, 21004]
Open Access
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
|
Open Access
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
|
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
Diamond Proposal Number(s):
[20223]
Open Access
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
|
|
E02-JEM ARM 300CF
|
Diamond Proposal Number(s):
[19064]
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
|
Diamond Proposal Number(s):
[19714]
Open Access
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
|
|
