Deriving De Novo Atomic Models by Cryo Electron Microscopy

Summary

Cryo electron microscopy (cryoEM) can be employed to derive de novo atomic models of macromolecular complexes in solution. The steps involved in high resolution cryoEM of biological molecules, from image recording, to data processing, to atomic modeling based on the resulting cryoEM density map, are illustrated.

Abstract

Cryo electron microscopy is a structural biology technique for determining three-dimensional structures of supramolecular complexes in solution. In cryoEM, samples in their physiological, non-crystalline state are embedded in vitreous ice during low dose imaging. A three-dimensional density map of the sample is obtained by combining tens of thousands of cryoEM images. In recent years, the resolution of cryoEM has improved steadily, reaching a resolution sufficient for building atomic models from the cryoEM density map alone.

In this video article, we illustrate how such atomic models can be derived by cryoEM. Others have described the detailed procedures for preparing frozen hydrated samples, image acquisition and the basic steps of data processing of cryoEM ^1,2. We will focus on several criticle technical aspects important for reaching 3-4 Å resolution and for building reliable atomic models. We use the tobacco mosaic virus (TMV) and cytoplasmic polyhedrosis virus (CPV) as representative complexes with helical and icosahedral objects, respectively. De novo atomic models of these objects have previously been determined by cryoEM and published, and their biological relevance is described in previous publications^3-6.

Introduction

Beginning in the early 1970s, electron microscopy has been used in structural biology to investigate macromolecular assemblies at a “biologically significant” resolution ⁷. Atomic resolution protein structures determined by x-ray crystallography or NMR were docked into the lower resolution (30-8 Å) electron microscopy density maps (for example^8,9). In this way, interactions between subunits of a large complex could be assessed to give hints at the overall function of a macromolecular assembly. While this approach is still frequently used for complexes with low or no symmetry, advances over the last twenty years in sample preparat....

Protocol

NOTE: A general workflow of atomic model determination by cryoEM is given in Fig. 1. In the following protocol, items 1, 2 and 4 are shared in both single particle- and helical-structural determination. Item 3.1 is for single particle structures while item 3.2 is for helical structures. Therefore, one either will follow 1, 2, 3.1, 4 or 1, 2, 3.2, 4 depending on the nature of the sample being studied.

1. Grid preparation, sample preparation, and image collection

1. Place commercial cry.......

Discussion

With advances in imaging hardware and software, cryoEM has come of age as a structural biology method enabling de novo atomic models based on density maps alone. However, some major limitations persist for this technique, in particular the limitations in sample types and preparations which are suitable for use in cryoEM. Thus when considering cryoEM for structural determination, the most important question to ask is still: is this the right technique for my sample ^15-17? While X-ray crystallography an.......

Materials

Name	Company	Catalog Number	Comments
Name of Equipment	Company	Catalog Number	Comments
Vitrobot	FEI
Titan Krios	FEI
Quantifoil cryoEM grids	Quantifoil microtools GmbH		http://www.quantifoil.com/qfstr_en.php4
1,2-dichloro-ethane	Sigma-Aldrich	107-06-2
SO163 Films and developer/fixer	Kodak
Super CoolScan 9000 ED	Nikon
EMAN 1,2	Baylor College of Medicine		http://blake.bcm.edu/emanwiki/EMAN/
Linux cluster with scientific linux (redhat enterprise linux)	Redhat / Fermilab
Chimera	UCSF		http://www.cgl.ucsf.edu/chimera/
Coot			http://lmb.bioch.ox.ac.uk/coot/
CNS			http://cns-online.org/v1.3/
Phenix			http://www.phenix-online.org/