Cryo-EM. Part A, Sample preparation and data collection / edited by Grant J. Jensen.

Format:

Book

Contributor:

Jensen, Grant J.

Series:

Methods in enzymology ; v. 481.

Methods in enzymology, 0076-6879 ; v. 481

Language:

English

Subjects (All):

Cryobiology--Methodology.

Cryobiology.

Electron microscopy--Methodology.

Electron microscopy.

Physical Description:

1 online resource (466 p.)

Edition:

1st ed.

Place of Publication:

San Diego, Calif. : Academic Press, an imprint of Elsevier, 2010.

Language Note:

English

Summary:

This volume is dedicated to a description of the instruments, samples, protocols, and analyses that belong to cryo-EM. It emphasizes the relatedness of the ideas, instrumentation, and methods underlying all cryo-EM approaches, which allow practitioners to easily move between them. Within each section, the articles are ordered according to the most common symmetry of the sample to which their methods are applied. * Includes time-tested core methods and new innovations applicable to any researcher * Methods included are useful to both established researchers and newcomers to th

Contents:

Front Cover; Methods in Enzymology Cryo-EM, Part A: Sample Preparation and Data Collection; Copyright Page; Contents; Contributors; Preface; Methods in Enzymology; Historical Perspective: 3D Reconstruction from Electron Micrographs: A Personal Account of its Development; Chapter 1: Preparation of 2D Crystals of Membrane Proteins for High-Resolution Electron Crystallography Data Collection; Abstract; 1. Introduction to Electron Crystallography; 2. Purification of Membrane Proteins; 3. 2D Crystallization of Membrane Proteins; 4. Requirements for Electron Crystallography Data Collection

5. ConclusionsReferences; Chapter 2: Helical Crystallization of Soluble and Membrane Binding Proteins; Abstract; 1. Introduction; 2. Materials and Methods; Acknowledgments; References; Chapter 3: Plunge Freezing for Electron Cryomicroscopy; Abstract; 1. Introduction; 2. Grids and Supports; 3. Cleaning the Grids; 4. Preparing the Cryogen; 5. Plunging the Grid; 6. Common Problems and Their Diagnoses; Acknowledgments; References; Chapter 4: A Practical Guide to the Use of Monolayer Purification and Affinity Grids; Abstract; 1. A Brief History of the Use of Lipid Monolayers in Electron Microscopy

2. Preparation of Lipid Monolayer Specimens3. Monolayer Purification; 4. Examples of Monolayer Purification Applications; 5. The Affinity Grid; 6. Examples of Affinity Grid Applications; 7. Characterization of Monolayer Specimens; 8. Challenges and Future Directions; Acknowledgments; References; Chapter 5: GraFix: Stabilization of Fragile Macromolecular Complexes for Single Particle Cryo-EM; Abstract; 1. Introduction; 2. Overview of the GraFix Procedure; 3. Methods; 4. Conclusions; References; Chapter 6: Cryonegative Staining of Macromolecular Assemblies; Abstract; 1. Introduction

2. Cryonegative Staining-The ``Adrian ́ ́ Method: Frozen-Hydrated Specimens in the Presence of a Saturated Ammonium Molybdate Solution at Neutral pH3. Cryonegative Staining with the Sandwich Method: The ``Holger Stark ́ ́ Alternative; 4. Conclusion; Acknowledgments; References; Chapter 7: Liposomes on a Streptavidin Crystal; Abstract; 1. Introduction; 2. Methods; 3. Conclusion; References; Chapter 8: Micromanipulator-Assisted Vitreous Cryosectioning and Sample Preparation by High-Pressure Freezing; Abstract; 1. Introduction; 2. Why is Vitreous Cryosectioning so Difficult?

3. High-Pressure Freezing for Vitreous Cryosectioning4. Extracellular Cryoprotectants for Vitreous Cryosectioning; 5. Mounting a Vitrified Sample in the Cryo-Ultramicrotome; 6. Hardware: Cryo-Ultramicrotomes; 7. The Micromanipulator; 8. Cryodiamond Knives; 9. The Cryotrimming Knife; 10. Setting up the Cryo-Ultramicrotome for Sectioning; 11. Trimming a Blockface; 12. Preparing to Cryosection; 13. The Active Static Ionizer; 14. Cryosectioning; 15. Transferring Cryosections to the EM Grid; 16. Pressing the Sections onto the Grid; 17. The Sectioning Environment and Relative Humidity

18. Cryosectioning Artifacts

Notes:

Description based upon print version of record.

Includes bibliographical references and indexes.

ISBN:

9786612878732

9781282878730

1282878735

9780080956954

0080956955

OCLC:

668081554