Workflow for High-resolution Sub-volume Averaging from Heterogenous Viral and Virus-like Assemblies

Sibert, Bryan; Kim, Joseph Y.; Yang, Jae E.; Hannon-Hatfield, Adam; Ke, Zunlong; Garfinkel, David; Wright, Elizabeth

doi:10.1093/micmic/ozad067.470

Cited by 5 publications

(3 citation statements)

References 14 publications

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“…Gaining structural insights from cryo-ET data is complex and remains an active area of research, with ongoing developments in CTF estimation 39 , tilt series alignment 8 , tomogram denoising 21,23,24,45,52 , automated localisation of structures of interest 30,[53][54][55][56][57][58][59][60][61][62] and approaches to deal with structural heterogeneity among extracted particles [63][64][65][66] , amongst many others. As a result, cryo-ET image processing pipelines, including the one described here, will need to be continuously updated in the coming years.…”

Section: Discussionmentioning

confidence: 99%

An image processing pipeline for electron cryo-tomography in RELION-5

Burt,

Toader,

Warshamanage

et al. 2024

Preprint

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Electron tomography of frozen, hydrated samples allows structure determination of macro-molecular complexes that are embedded in complex environments. Provided that the target complexes may be localised in noisy, three-dimensional tomographic reconstructions, averaging images of multiple instances of these molecules can lead to structures with sufficient resolution for de novo atomic modelling. Although many research groups have contributed image processing tools for these tasks, a lack of standardisation and inter-operability represents a barrier for newcomers to the field. Here, we present an image processing pipeline for electron tomography data in RELION-5, with functionality ranging from the import of unprocessed movies to the automated building of atomic models in the final maps. Our explicit definition of metadata items that describe the steps of our pipeline has been designed for inter-operability with other software tools and provides a framework for further standardisation.

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Section: Discussionmentioning

confidence: 99%

An image processing pipeline for electron cryo-tomography in RELION-5

Burt,

Toader,

Warshamanage

et al. 2024

Preprint

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“…This methodology adheres to a standardized and well-established workflow supported by advanced software packages such as Relion 1 and cryoSparc 2 . In parallel, cryogenic electron tomography (cryoET), coupled with subtomogram averaging (STA), expands the investigative scope to encompass heterogeneous macromolecules in their native context [3][4][5][6][7][8][9][10] . To enhance the resolution of subunits within in situ macromolecules, subtomograms (i.e., particles) are extracted from each tomogram and then subjected to 3D alignment and averaging, thereby improving signal-to-noise ratio.…”

Section: Introductionmentioning

confidence: 99%

TomoNet: A streamlined cryoET software pipeline with automatic particle picking on flexible lattices

Wang,

Liao,

et al. 2024

Preprint

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Cryogenic electron tomography (cryoET) is capable of determining in situ biological structures of molecular complexes at near atomic resolution by averaging half a million subtomograms. While abundant complexes/particles are often clustered in arrays, precisely locating and seamlessly averaging such particles across many tomograms present major challenges. Here, we developed TomoNet, a software package with a modern graphical user interface to carry out the entire pipeline of cryoET and subtomogram averaging to achieve high resolution. TomoNet features built-in automatic particle picking and 3D classification functions and integrates commonly used packages to streamline high-resolution subtomogram averaging for structures in one-, two- or three-dimensional arrays. Automatic particle picking is accomplished in two complementary ways: one based on template matching and the other employing deep learning. TomoNet's hierarchical file organization and visual display facilitate efficient data management as required for large cryoET datasets. Applications of TomoNet to three types of datasets demonstrate its capability of efficient and accurate particle picking on flexible and imperfect lattices to obtain high-resolution 3D biological structures: virus-like particles, bacterial surface layers within cellular lamellae, and membranes decorated with nuclear egress protein complexes. These results demonstrate TomoNet's potential for broad applications to various cryoET projects targeting high-resolution in situ structures.

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“…2 In parallel, cryogenic electron tomography (cryoET), coupled with subtomogram averaging (STA), expands the investigative scope to encompass heterogeneous macromolecules in their native context. [3][4][5][6][7][8][9][10] To enhance the resolution of subunits within in situ macromolecules, subtomograms (i.e., particles) are extracted from each tomogram and then subjected to three-dimensional (3D) alignment and averaging, thereby improving signal-to-noise ratio. Notably, STA has achieved resolutions up to sub-3 Å for in situ structures of large cellular complexes such as ribosomes, approaching the capabilities of single-particle cryoEM methodologies.…”

Section: Introductionmentioning

confidence: 99%

TomoNet: A streamlined cryogenic electron tomography software pipeline with automatic particle picking on flexible lattices

Wang,

Liao,

et al. 2024

Biol. Imaging

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Cryogenic electron tomography (cryoET) is capable of determining in situ biological structures of molecular complexes at near-atomic resolution by averaging half a million subtomograms. While abundant complexes/particles are often clustered in arrays, precisely locating and seamlessly averaging such particles across many tomograms present major challenges. Here, we developed TomoNet, a software package with a modern graphical user interface to carry out the entire pipeline of cryoET and subtomogram averaging to achieve high resolution. TomoNet features built-in automatic particle picking and three-dimensional (3D) classification functions and integrates commonly used packages to streamline high-resolution subtomogram averaging for structures in 1D, 2D, or 3D arrays. Automatic particle picking is accomplished in two complementary ways: one based on template matching and the other using deep learning. TomoNet's hierarchical file organization and visual display facilitate efficient data management as required for large cryoET datasets. Applications of TomoNet to three types of datasets demonstrate its capability of efficient and accurate particle picking on flexible and imperfect lattices to obtain high-resolution 3D biological structures: virus-like particles, bacterial surface layers within cellular lamellae, and membranes decorated with nuclear egress protein complexes. These results demonstrate TomoNet's potential for broad applications to various cryoET projects targeting high-resolution in situ structures. Impact StatementCryogenic electron tomography (cryoET) has become a powerful approach to visualize the organization and high-resolution structures of biological complexes in their native environment. Subtomogram averaging (STA) of hundreds of thousands of particles (i.e., subtomograms) is necessary to obtain near-atomic resolution structures for each such complex. While abundant biological complexes often cluster in arrays that manifest as one to three-dimensional lattices, flexibility and imperfection of such lattices pose challenges for efficient and accurate particle picking. To overcome these challenges and to meet the growing demand for efficient data processing and management in the cryoET and STA workflow, we have developed TomoNet, a user-friendly software package with a modern graphical user interface that allows users to execute the entire data processing pipeline seamlessly with the integration of commonly used software packages. TomoNet addresses the particlepicking challenge with two solutions, one based on geometric template matching and the other using artificial intelligence. Applications of TomoNet to three representative datasets demonstrate its capability for highresolution structure determination of biological complexes on flexible and imperfect lattices.

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Workflow for High-resolution Sub-volume Averaging from Heterogenous Viral and Virus-like Assemblies

Cited by 5 publications

References 14 publications

An image processing pipeline for electron cryo-tomography in RELION-5

An image processing pipeline for electron cryo-tomography in RELION-5

TomoNet: A streamlined cryoET software pipeline with automatic particle picking on flexible lattices

TomoNet: A streamlined cryogenic electron tomography software pipeline with automatic particle picking on flexible lattices

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