Jensen Cryo-EM Part A: Sample Preparation and Data Collection

1. Auflage 2010
ISBN: 978-0-08-095695-4
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 481, 464 Seiten

Reihe: Methods in Enzymology

Cryo-EM Part A: Sample Preparation and Data Collection 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 the field - Relevant background and reference information given for procedures can be used as a guide

Autoren/Hrsg.

Jensen, Grant

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Enzymology Cryo-EM, Part A: Sample Preparation and Data Collection;4
3;Copyright Page;5
4;Contents;6
5;Contributors;12
6;Preface;16
7;Methods in Enzymology;18
8;Historical Perspective: 3D Reconstruction from Electron Micrographs: A Personal Account of its Development;47
9;Chapter 1: Preparation of 2D Crystals of Membrane Proteins for High-Resolution Electron Crystallography Data Collection;71
9.1;Abstract;71
9.2;1. Introduction to Electron Crystallography;72
9.3;2. Purification of Membrane Proteins;72
9.4;3. 2D Crystallization of Membrane Proteins;74
9.5;4. Requirements for Electron Crystallography Data Collection;78
9.6;5. Conclusions;86
9.7;References;87
10;Chapter 2: Helical Crystallization of Soluble and Membrane Binding Proteins;91
10.1;Abstract;91
10.2;1. Introduction;92
10.3;2. Materials and Methods;95
10.4;Acknowledgments;104
10.5;References;104
11;Chapter 3: Plunge Freezing for Electron Cryomicroscopy;109
11.1;Abstract;109
11.2;1. Introduction;110
11.3;2. Grids and Supports;111
11.4;3. Cleaning the Grids;112
11.5;4. Preparing the Cryogen;114
11.6;5. Plunging the Grid;117
11.7;6. Common Problems and Their Diagnoses;123
11.8;Acknowledgments;124
11.9;References;126
12;Chapter 4: A Practical Guide to the Use of Monolayer Purification and Affinity Grids;129
12.1;Abstract;130
12.2;1. A Brief History of the Use of Lipid Monolayers in Electron Microscopy;131
12.3;2. Preparation of Lipid Monolayer Specimens;132
12.4;3. Monolayer Purification;135
12.5;4. Examples of Monolayer Purification Applications;138
12.6;5. The Affinity Grid;140
12.7;6. Examples of Affinity Grid Applications;144
12.8;7. Characterization of Monolayer Specimens;148
12.9;8. Challenges and Future Directions;150
12.10;Acknowledgments;151
12.11;References;151
13;Chapter 5: GraFix: Stabilization of Fragile Macromolecular Complexes for Single Particle Cryo-EM;155
13.1;Abstract;155
13.2;1. Introduction;156
13.3;2. Overview of the GraFix Procedure;158
13.4;3. Methods;166
13.5;4. Conclusions;171
13.6;References;171
14;Chapter 6: Cryonegative Staining of Macromolecular Assemblies;173
14.1;Abstract;173
14.2;1. Introduction;174
14.3;2. Cryonegative Staining-The ``Adrian´´ Method: Frozen-Hydrated Specimens in the Presence of a Saturated Ammonium Molybdate Solution at Neutral pH;179
14.4;3. Cryonegative Staining with the Sandwich Method: The ``Holger Stark´´ Alternative;185
14.5;4. Conclusion;187
14.6;Acknowledgments;189
14.7;References;189
15;Chapter 7: Liposomes on a Streptavidin Crystal;193
15.1;Abstract;193
15.2;1. Introduction;194
15.3;2. Methods;195
15.4;3. Conclusion;208
15.5;References;209
16;Chapter 8: Micromanipulator-Assisted Vitreous Cryosectioning and Sample Preparation by High-Pressure Freezing;211
16.1;Abstract;211
16.2;1. Introduction;212
16.3;2. Why is Vitreous Cryosectioning so Difficult?;213
16.4;3. High-Pressure Freezing for Vitreous Cryosectioning;214
16.5;4. Extracellular Cryoprotectants for Vitreous Cryosectioning;216
16.6;5. Mounting a Vitrified Sample in the Cryo-Ultramicrotome;217
16.7;6. Hardware: Cryo-Ultramicrotomes;219
16.8;7. The Micromanipulator;222
16.9;8. Cryodiamond Knives;223
16.10;9. The Cryotrimming Knife;225
16.11;10. Setting up the Cryo-Ultramicrotome for Sectioning;225
16.12;11. Trimming a Blockface;226
16.13;12. Preparing to Cryosection;228
16.14;13. The Active Static Ionizer;229
16.15;14. Cryosectioning;230
16.16;15. Transferring Cryosections to the EM Grid;232
16.17;16. Pressing the Sections onto the Grid;233
16.18;17. The Sectioning Environment and Relative Humidity;235
16.19;18. Cryosectioning Artifacts;237
16.20;19. Conclusion;238
16.21;Acknowledgments;239
16.22;References;239
17;Chapter 9: Site-Specific Biomolecule Labeling with Gold Clusters;241
17.1;Abstract;241
17.2;1. Introduction;242
17.3;2. General Considerations;247
17.4;3. Monolayer Protected Cluster Labeling of Biomolecules;249
17.5;4. Nanogold Labeling of Proteins;258
17.6;5. Ni(II)-NTA-Nanogold Labeling of His-Tagged Proteins;268
17.7;6. Conclusions;271
17.8;References;271
18;Chapter 10: How to Operate a Cryo-Electron Microscope;277
18.1;Abstract;277
18.2;1. Introduction;278
18.3;2. Basic Operations of Cryo-EM;279
18.4;3. Several Practicals on Imaging Frozen-Hydrated Biological Specimens;289
18.5;4. Concluding Remarks;294
18.6;References;295
19;Chapter 11: Collecting Electron Crystallographic Data of Two-Dimensional Protein Crystals;297
19.1;Abstract;297
19.2;1. Introduction;298
19.3;2. Challenges;299
19.4;3. Images and Diffraction Patterns;301
19.5;4. Specimen Preparation;303
19.6;5. Specimen Flatness;310
19.7;6. Data Collection;312
19.8;Acknowledgments;326
19.9;References;326
20;Chapter 12: Automated Data Collection for Electron Microscopic Tomography;329
20.1;Abstract;329
20.2;1. Introduction;330
20.3;2. Predictive EMT Data Collection;333
20.4;3. Real-Time Tomographic Reconstruction;343
20.5;4. Dual-Axis EMT Data Collection;349
20.6;5. Supporting Automation Procedures;355
20.7;6. Summary;358
20.8;Acknowledgments;359
20.9;References;359
21;Chapter 13: Correlated Light and Electron Cryo-Microscopy;363
21.1;Abstract;363
21.2;1. Introduction;363
21.3;2. Correlative FLM/ECM with Freezing after FLM Imaging;365
21.4;3. Correlative FLM and ECM, with Freezing before FLM Imaging;368
21.5;Acknowledgments;386
21.6;References;386
22;Chapter 14: Phase Plates for Transmission Electron Microscopy;389
22.1;Abstract;390
22.2;1. Introduction;390
22.3;2. Types of Phase Plates and Historical Notes;393
22.4;3. Microscope Requirements and Modifications;402
22.5;4. Phase Plate Operation Procedures;405
22.6;5. Summary and Future Prospects;412
22.7;Acknowledgments;413
22.8;References;413
23;Chapter 15: Radiation Damage in Electron Cryomicroscopy;417
23.1;Abstract;417
23.2;1. Introduction;418
23.3;2. Measuring Electron Exposure;418
23.4;3. Energy Absorbed by Specimens;420
23.5;4. Radiation Damage and Choice of Accelerating Voltage;420
23.6;5. Primary and Secondary Damage to Proteins During Irradiation;423
23.7;6. Tertiary Damage to Proteins During Irradiation;424
23.8;7. Cryoprotection and Optimal Temperatures;425
23.9;8. Quantification of Beam Damage with Increasing Exposure;427
23.10;9. Optimal Exposures for Thin Crystals;429
23.11;10. Optimal Exposures for Single Particle Samples;430
23.12;11. Optimal Exposures for Tomographic Samples;432
23.13;12. Concluding Comments;432
23.14;Acknowledgments;433
23.15;References;433
23.16;Author Index;435
23.17;Subject Index;447
23.18;Colorplate;457