Molecular Electron Microscopy in Neuroscience: An Approach to Study Macromolecular Assemblies

2015 ◽  
pp. 205-216 ◽  
Author(s):  
Bjoern Sander ◽  
Monika M. Golas
Keyword(s):  
Electron Microscopy ◽  
Macromolecular Assemblies ◽  
Molecular Electron

scholarly journals Improved metrics for comparing structures of macromolecular assemblies determined by 3D electron-microscopy

2017 ◽  
Vol 199 (1) ◽  
pp. 12-26 ◽  
Author(s):  
Agnel Praveen Joseph ◽  
Ingvar Lagerstedt ◽  
Ardan Patwardhan ◽  
Maya Topf ◽  
Martyn Winn
Keyword(s):  
Electron Microscopy ◽  
Macromolecular Assemblies ◽  
3D Electron Microscopy ◽  
3D Electron

Frontiers in Cryo Electron Microscopy of Complex Macromolecular Assemblies

2019 ◽  
Vol 21 (1) ◽  
pp. 395-415 ◽  
Author(s):  
Jana Ognjenović ◽  
Reinhard Grisshammer ◽  
Sriram Subramaniam
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Specimen Preparation ◽  
Macromolecular Assemblies ◽  
Cryo Electron Microscopy ◽  
G Protein Coupled ◽  
Improved Methods

In recent years, cryo electron microscopy (cryo-EM) technology has been transformed with the development of better instrumentation, direct electron detectors, improved methods for specimen preparation, and improved software for data analysis. Analyses using single-particle cryo-EM methods have enabled determination of structures of proteins with sizes smaller than 100 kDa and resolutions of ∼2 Å in some cases. The use of electron tomography combined with subvolume averaging is beginning to allow the visualization of macromolecular complexes in their native environment in unprecedented detail. As a result of these advances, solutions to many intractable challenges in structural and cell biology, such as analysis of highly dynamic soluble and membrane-embedded protein complexes or partially ordered protein aggregates, are now within reach. Recent reports of structural studies of G protein–coupled receptors, spliceosomes, and fibrillar specimens illustrate the progress that has been made using cryo-EM methods, and are the main focus of this review.

Protein Characterization by Low-Angle, Rotary-Replication Electron Microscopy

2000 ◽  
Vol 6 (S2) ◽  
pp. 994-995
Author(s):  
S. Samuelsson
Keyword(s):  
Electron Microscopy ◽  
Heavy Metals ◽  
Freeze Fracture ◽  
Accurate Method ◽  
Protein Characterization ◽  
Wild Type ◽  
Macromolecular Assemblies ◽  
Transmission Electron ◽  
Excellent Method ◽  
Atomically Flat

There are several established strategies for visualizing proteins by transmission electron microscopy (TEM). These include negative staining, cryo-TEM, glycerol-spray/rotary-replication, mica-flake/freeze-fracture. Of these, low-angle, rotary-replication of proteins dried out of glycerol has proven to be a reliable and accurate method for studying surface topologies of macromolecular assemblies and particles. We have characterized many proteins, both chimeras and wild type; methods, caveats and the surface structure of a couple examples are described here.The technique of using heavy metals to cast replicas of proteins and particles has been around for decades (metal replication of proteins was first described in 1956) and continues to provide an excellent method for evaluating protein topology. The success of this method is based on the physical properties of protein in contact with freshly cleaved mica, glycerol and atomic platinum. Mica is easily split and atomically flat making it an excellent substrate.

scholarly journals Single-particle reconstruction of biological macromolecules in electron microscopy – 30 years

2009 ◽  
Vol 42 (3) ◽  
pp. 139-158 ◽  
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Specimen Preparation ◽  
Biological Macromolecules ◽  
Personal Account ◽  
Dynamic Information ◽  
Preparation Methods ◽  
Single Particle Reconstruction ◽  
Macromolecular Assemblies ◽  
Particle Reconstruction

AbstractThis essay gives the autho's personal account on the development of concepts underlying single-particle reconstruction, a technique in electron microscopy of macromolecular assemblies with a remarkable record of achievements as of late. The ribosome proved to be an ideal testing ground for the development of specimen preparation methods, cryo-EM techniques, and algorithms, with discoveries along the way as a rich reward. Increasingly, cryo-EM and single-particle reconstruction, in combination with classification techniques, is revealing dynamic information on functional molecular machines uninhibited by molecular contacts.

scholarly journals Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies

2014 ◽  
Vol 187 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Guimei Yu ◽  
Frank Vago ◽  
Dongsheng Zhang ◽  
Jonathan E. Snyder ◽  
Rui Yan ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Structural Analysis ◽  
Single Step ◽  
Macromolecular Assemblies ◽  
Cryo Electron Microscopy

scholarly journals Automatic whole cell organelle segmentation in volumetric electron microscopy

2020 ◽  
Author(s):  
Larissa Heinrich ◽  
Davis Bennett ◽  
David Ackerman ◽  
Woohyun Park ◽  
John Bogovic ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Open Data ◽  
Computer Code ◽  
Cell Types ◽  
Cell Organelle ◽  
Whole Cells ◽  
Macromolecular Assemblies

Cells contain hundreds of different organelle and macromolecular assemblies intricately organized relative to each other to meet any cellular demands. Obtaining a complete understanding of their organization is challenging and requires nanometer-level, threedimensional reconstruction of whole cells. Even then, the immense size of datasets and large number of structures to be characterized requires generalizable, automatic methods. To meet this challenge, we developed an analysis pipeline for comprehensively reconstructing and analyzing the cellular organelles in entire cells imaged by focused ion beam scanning electron microscopy (FIB-SEM) at a near-isotropic size of 4 or 8 nm per voxel. The pipeline involved deep learning architectures trained on diverse samples for automatic reconstruction of 35 different cellular organelle classes - ranging from endoplasmic reticulum to microtubules to ribosomes - from multiple cell types.Automatic reconstructions were used to directly quantify various previously inaccessible metrics about these structures, including their spatial interactions. We show that automatic organelle reconstructions can also be used to automatically register light and electron microscopy images for correlative studies. We created an open data and open source web repository, OpenOrganelle, to share the data, computer code, and trained models, enabling scientists everywhere to query and further reconstruct the datasets.

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