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Guidelines for using Bsoft for high resolution reconstruction and validation of biomolecular structures from electron micrographs.

ABSTRACT: Cryo-electron microscopy (cryoEM) is becoming popular as a tool to solve biomolecular structures with the recent availability of direct electron detectors allowing automated acquisition of high resolution data. The Bsoft software package, developed over 20 years for analyzing electron micrographs, offers a full workflow for validated single particle analysis with extensive functionality, enabling customization for specific cases. With the increasing use of cryoEM and its automation, proper validation of the results is a bigger concern. The three major validation approaches, independent data sets, resolution-limited processing, and coherence testing, can be incorporated into any Bsoft workflow. Here, the main workflow is divided into four phases: (i) micrograph preprocessing, (ii) particle picking, (iii) particle alignment and reconstruction, and (iv) interpretation. Each of these phases represents a conceptual unit that can be automated, followed by a check point to assess the results. The aim in the first three phases is to reconstruct one or more validated maps at the best resolution possible. Map interpretation then involves identification of components, segmentation, quantification, and modeling. The algorithms in Bsoft are well established, with future plans focused on ease of use, automation and institutionalizing validation.

SUBMITTER: Heymann JB

PROVIDER: S-EPMC5734390 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Guidelines for using Bsoft for high resolution reconstruction and validation of biomolecular structures from electron micrographs.

Heymann J Bernard JB

Protein science : a publication of the Protein Society 20171009 1

Cryo-electron microscopy (cryoEM) is becoming popular as a tool to solve biomolecular structures with the recent availability of direct electron detectors allowing automated acquisition of high resolution data. The Bsoft software package, developed over 20 years for analyzing electron micrographs, offers a full workflow for validated single particle analysis with extensive functionality, enabling customization for specific cases. With the increasing use of cryoEM and its automation, proper valid ...[more]

PMID: 28891250

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Project description:Scanning electron microscopy (SEM) provides a technical platform for nanoscopic mapping of biological structures. Correct preparation of SEM samples can provide an unprecedented understanding of the nexus between cellular morphology and topography. This comparative study critically examines two coating methods for preparing biological samples for scanning electron microscopy, while also providing novel advice on how to prepare in vitro epithelial or endothelial samples for high-resolution scanning-electron microscopy (HR-SEM). Two obstacles often confront the biologist when investigating cellular structures grown under tissue culture conditions, namely., how to prepare and present the biological samples to the HR-SEM microscope without affecting topographical membrane and cellular structural alterations. Firstly, our use of the Millicell cellulose inserts on which to grow our cellular samples in preparation for HR-SEM is both novel and advantageous to comparing the permeability function of cells to their morphological function. Secondly, biological material is often non-conducting, thermally sensitive and fragile and, therefore, needs to be fixed correctly and coated with thin conducting metal to ensure high-resolution detail of samples. Immortalized mouse brain endothelial cells (bEnd5) was used as a basis for describing the preferences in the use of the protocol. We compare two biological sample coating modalities for the visualizing and analysis of texturized, topographical, membranous ultrastructures of brain endothelial cell (BEC) confluent monolayers, namely, carbon and gold:palladium (Au:Pd) sputter coating in preparation for HR-SEM. BEC monolayers sputter-coated with these two modalities produced three-dimensional micrographs which have distinctly different topographical detail from which the nanostructural cellular data can be examined. The two coating methods display differences in the amount of nanoscopic detail that could be resolved in the nanosized membrane cytoarchitecture of BEC monolayers. The micrographical data clearly showed that Au:Pd sputter-coated samples generate descript imagery, providing useful information for profiling membrane nanostructures compared to carbon-coated samples. The recommendations regarding the contrast in two modalities would provide the necessary guidance to biological microscopists in preparing tissue culture samples for HR-SEM.

Dataset Information

Guidelines for using Bsoft for high resolution reconstruction and validation of biomolecular structures from electron micrographs.

Publications

Guidelines for using Bsoft for high resolution reconstruction and validation of biomolecular structures from electron micrographs.

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