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      Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces

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          Abstract

          Over the last few years, cryo electron microscopy has become the most important method in structural biology. While 80% of deposited maps are from single particle analysis, electron tomography has grown to become the second most important method. In particular sub-tomogram averaging has matured as a method, delivering structures between 2 and 5 Å from complexes in cells as well as in vitro complexes. While this resolution range is not standard, novel developments point toward a promising future. Here, we provide a guide for the workflow from sample to structure to gain insight into this emerging field.

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          CTFFIND4: Fast and accurate defocus estimation from electron micrographs.

          CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope's contrast transfer function (CTF) to an image's amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled.
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            Automated electron microscope tomography using robust prediction of specimen movements.

            A new method was developed to acquire images automatically at a series of specimen tilts, as required for tomographic reconstruction. The method uses changes in specimen position at previous tilt angles to predict the position at the current tilt angle. Actual measurement of the position or focus is skipped if the statistical error of the prediction is low enough. This method allows a tilt series to be acquired rapidly when conditions are good but falls back toward the traditional approach of taking focusing and tracking images when necessary. The method has been implemented in a program, SerialEM, that provides an efficient environment for data acquisition. This program includes control of an energy filter as well as a low-dose imaging mode, in which tracking and focusing occur away from the area of interest. The program can automatically acquire a montage of overlapping frames, allowing tomography of areas larger than the field of the CCD camera. It also includes tools for navigating between specimen positions and finding regions of interest.
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              Computer visualization of three-dimensional image data using IMOD.

              We have developed a computer software package, IMOD, as a tool for analyzing and viewing three-dimensional biological image data. IMOD is useful for studying and modeling data from tomographic, serial section, and optical section reconstructions. The software allows image data to be visualized by several different methods. Models of the image data can be visualized by volume or contour surface rendering and can yield quantitative information.
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                Author and article information

                Contributors
                Role: Academic Editor
                Role: Academic Editor
                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                08 June 2021
                June 2021
                : 22
                : 12
                : 6177
                Affiliations
                Department of Biomedical Engineering and Health Systems, Royal Technical Institute (KTH), Hälsovägen 11C, 141 27 Huddinge, Sweden; zuonewan@ 123456kth.se (Z.W.); qinzha@ 123456kth.se (Q.Z.)
                Author notes
                [* ]Correspondence: carmim@ 123456kth.se
                [†]

                These authors contributed equally.

                Author information
                https://orcid.org/0000-0002-5530-0815
                https://orcid.org/0000-0001-6402-8270
                Article
                ijms-22-06177
                10.3390/ijms22126177
                8228724
                e3646410-61c1-4f4f-a8a2-7e5a5adee1d2
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 06 May 2021
                : 02 June 2021
                Categories
                Review

                Molecular biology
                protein complexes,structural biology,electron microscopy
                Molecular biology
                protein complexes, structural biology, electron microscopy

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