Technical Advances in Cryo-Electron Microscopy

Editorial

22 August 2019

Editorial: Technical Advances in Cryo-Electron Microscopy

Angel Rivera-Calzada

and

Marta Carroni

9,522 views

14 citations

Editors

Marta Carroni

Science for Life Laboratory (SciLifeLab)

Angel Rivera-Calzada

Spanish National Cancer Research Center

Impact

(A–C) Cryo-EM images are usually acquired on heterogeneous samples. Contaminants (light blue), denatured or partially disassemble complexes (light red) as well as different protein complex conformations (gold) are imaged together with the specimen (gray). (D) General approach workflow to handle sample heterogeneity during image processing. Processes (black boxes), iterative process (green boxes), and specific input data (blue boxes) are indicated. The consensus reconstruction is obtained from an initially heterogeneous dataset and it will be used as the initial reference for more specific refinement protocols to deal with sample heterogeneity.

Mini Review

15 May 2019

Hands on Methods for High Resolution Cryo-Electron Microscopy Structures of Heterogeneous Macromolecular Complexes

Marina Serna

Electron microscopy of frozen hydrated samples (cryo-EM) is a powerful structural technique that allows the direct study of functional macromolecular complexes in an almost physiological environment. Protein macromolecular complexes are dynamic structures that usually hold together by an intricate network of protein-protein interactions that can be weak and transient. Moreover, a standard feature of many of these complexes is that they behave as nanomachines able to undergo functionally relevant conformational changes in one or several complex components. Among all the other main structural biology techniques, only cryo-EM has the potential of successfully dealing at the same time with both sample heterogeneity and inherent flexibility. The cryo-EM field is currently undergoing a revolution thanks to groundbreaking technical developments that have brought within our reach the possibility of solving the structure of biological complexes at atomic resolution. These technical developments have been mostly focused on new direct electron detector technology and improved sample preparation methods leading to better image quality. This fact has in turn required the development of new and better image processing algorithms to make the most of the higher quality data. The aim of this review is to provide a brief overview of some reported examples of single particle analysis strategies designed to find different conformational and compositional states within target macromolecular complex and specifically to deal with it to reach higher resolution information. Different image processing methodologies specifically aimed to symmetric or pseudo-symmetric protein complexes will also be discussed.

34,622 views

46 citations

Protocols

04 September 2018

Protocols for Subtomogram Averaging of Membrane Proteins in the Dynamo Software Package

Paula P. Navarro

, 1 more and

Daniel Castaño-Díez

Cryo-electron tomography allows low-resolution three-dimensional (3D) viewing of cellular organelles and macromolecular complexes present as multiple copies within a tomogram. These structures are computationally extracted and averaged in order to obtain high-resolution 3D structures, and provide a map of their spatial distribution and interaction with their biological microenvironment. To do so, we apply the user-friendly Dynamo software package on a tomographic data set. Dynamo acts as a modular toolbox adaptable to different biological scenarios, allowing a custom designed pipeline for subtomogram averaging. Here, we use as a textbook example the mitochondrial docking site of the positive-strand RNA Flock house nodavirus (FHV) to describe how Dynamo coordinates several basic steps in the subtomogram averaging workflow. Our framework covers specific strategies to deal with additional issues in subtomogram averaging as tomographic data management, 3D surface visualization, automatic assignment of asymmetry and inherent loss of Fourier information in presence of preferential views.

12,365 views

36 citations

The plots of sweep number vs. (A) intensity of average per spot and (B) total number of measurable reflection numbers in the different resolution sphere shells of merged 3D reciprocal lattice, each resolution bin was colored differently.

Mini Review

12 December 2018

The Evolution and the Advantages of MicroED

Brent L. Nannenga

, 1 more and

Dan Shi

10,292 views

17 citations

Mini Review

31 July 2018

Cryo-EM Grid Preparation of Membrane Protein Samples for Single Particle Analysis

Germán G. Sgro

and

Tiago R. D. Costa

Recent advances in cryo-electron microscopy (cryo-EM) have made it possible to solve structures of biological macromolecules at near atomic resolution. Development of more stable microscopes, improved direct electron detectors and faster software for image processing has enabled structural solution of not only large macromolecular (megadalton range) complexes but also small (~60 kDa) proteins. As a result of the widespread use of the technique, we have also witnessed new developments of techniques for cryo-EM grid preparation of membrane protein samples. This includes new types of solubilization strategies that better stabilize these protein complexes and the development of new grid supports with proven efficacy in reducing the motion of the molecules during electron beam exposure. Here, we discuss the practicalities and recent challenges of membrane protein sample preparation and vitrification, as well as grid support and foil treatment in the context of the structure determination of protein complexes by single particle cryo-EM.

Generic protein purification workflow and different membrane protein stabilization strategies using artificial membranes. (A) Cytoplasmic or membrane proteins are initially expressed in liquid or solid cultures, and pellets are stored after harvesting by centrifugation. Different physical or chemical cell disruption methods are utilized for releasing cytoplasmic proteins into solution or to obtain cell membrane extracts. Impure cytoplasmic proteins or solubilized cell membranes containing the protein of interest are purified by combination of different fast protein liquid chromatography (FPLC) methods. After protein stability, integrity and activity is verified by various biophysical techniques. The final sample concentration and buffer composition are adjusted before EM grid preparation. (B) Protein transmembrane domains are protected by the hydrophobic cell membrane phospholipid acyl chains. Micelles are spherical vesicles in which the detergent hydrophobic chains face inward and the hydrophilic polar heads face outward. Bicelles are obtained by a mixture of lipids and short chain detergents. The lipids will interact with the protein to form a lipid bilayer and the detergent will form the rim of the bicelle. Micelles will form after the solubilization of the membrane protein by detergents. SMALP (styrene-maleic acid lipid particles) are polymeric nanoparticles that protect the acyl chain of the lipid bilayer. Nanodiscs are lipid bilayers stabilized by wrapping a belt of amphipathic helix-rich membrane scaffold proteins (MSPs) around the detergent-solubilized membrane proteins. Amphipol polymers wrap around the hydrophobic patches of the membrane protein to form a stable complex in solution. Liposomes are artificial spherical lipid membranes where membrane proteins can assemble.

39,900 views

27 citations

Protocols

04 June 2018

Benchmarking cryo-EM Single Particle Analysis Workflow

Laura Y. Kim

, 8 more and

Bridget Carragher

Cryo electron microscopy facilities running multiple instruments and serving users with varying skill levels need a robust and reliable method for benchmarking both the hardware and software components of their single particle analysis workflow. The workflow is complex, with many bottlenecks existing at the specimen preparation, data collection and image analysis steps; the samples and grid preparation can be of unpredictable quality, there are many different protocols for microscope and camera settings, and there is a myriad of software programs for analysis that can depend on dozens of settings chosen by the user. For this reason, we believe it is important to benchmark the entire workflow, using a standard sample and standard operating procedures, on a regular basis. This provides confidence that all aspects of the pipeline are capable of producing maps to high resolution. Here we describe benchmarking procedures using a test sample, rabbit muscle aldolase.