Cryo-EM Part A: Sample Preparation and Data Collection (eBook)

Front Cover 1
Methods in Enzymology Cryo-EM, Part A: Sample Preparation and Data Collection 4
Copyright Page 5
Contents 6
Contributors 12
Preface 16
Methods in Enzymology 18
Historical Perspective: 3D Reconstruction from Electron Micrographs: A Personal Account of its Development 47
Chapter 1: Preparation of 2D Crystals of Membrane Proteins for High-Resolution Electron Crystallography Data Collection 71
Abstract 71
1. Introduction to Electron Crystallography 72
2. Purification of Membrane Proteins 72
3. 2D Crystallization of Membrane Proteins 74
4. Requirements for Electron Crystallography Data Collection 78
5. Conclusions 86
References 87
Chapter 2: Helical Crystallization of Soluble and Membrane Binding Proteins 91
Abstract 91
1. Introduction 92
2. Materials and Methods 95
Acknowledgments 104
References 104
Chapter 3: Plunge Freezing for Electron Cryomicroscopy 109
Abstract 109
1. Introduction 110
2. Grids and Supports 111
3. Cleaning the Grids 112
4. Preparing the Cryogen 114
5. Plunging the Grid 117
6. Common Problems and Their Diagnoses 123
Acknowledgments 124
References 126
Chapter 4: A Practical Guide to the Use of Monolayer Purification and Affinity Grids 129
Abstract 130
1. A Brief History of the Use of Lipid Monolayers in Electron Microscopy 131
2. Preparation of Lipid Monolayer Specimens 132
3. Monolayer Purification 135
4. Examples of Monolayer Purification Applications 138
5. The Affinity Grid 140
6. Examples of Affinity Grid Applications 144
7. Characterization of Monolayer Specimens 148
8. Challenges and Future Directions 150
Acknowledgments 151
References 151
Chapter 5: GraFix: Stabilization of Fragile Macromolecular Complexes for Single Particle Cryo-EM 155
Abstract 155
1. Introduction 156
2. Overview of the GraFix Procedure 158
3. Methods 166
4. Conclusions 171
References 171
Chapter 6: Cryonegative Staining of Macromolecular Assemblies 173
Abstract 173
1. Introduction 174
2. Cryonegative Staining-The ``Adrian´´ Method: Frozen-Hydrated Specimens in the Presence of a Saturated Ammonium Molybdate Solution at Neutral pH 179
3. Cryonegative Staining with the Sandwich Method: The ``Holger Stark´´ Alternative 185
4. Conclusion 187
Acknowledgments 189
References 189
Chapter 7: Liposomes on a Streptavidin Crystal 193
Abstract 193
1. Introduction 194
2. Methods 195
3. Conclusion 208
References 209
Chapter 8: Micromanipulator-Assisted Vitreous Cryosectioning and Sample Preparation by High-Pressure Freezing 211
Abstract 211
1. Introduction 212
2. Why is Vitreous Cryosectioning so Difficult? 213
3. High-Pressure Freezing for Vitreous Cryosectioning 214
4. Extracellular Cryoprotectants for Vitreous Cryosectioning 216
5. Mounting a Vitrified Sample in the Cryo-Ultramicrotome 217
6. Hardware: Cryo-Ultramicrotomes 219
7. The Micromanipulator 222
8. Cryodiamond Knives 223
9. The Cryotrimming Knife 225
10. Setting up the Cryo-Ultramicrotome for Sectioning 225
11. Trimming a Blockface 226
12. Preparing to Cryosection 228
13. The Active Static Ionizer 229
14. Cryosectioning 230
15. Transferring Cryosections to the EM Grid 232
16. Pressing the Sections onto the Grid 233
17. The Sectioning Environment and Relative Humidity 235
18. Cryosectioning Artifacts 237
19. Conclusion 238
Acknowledgments 239
References 239
Chapter 9: Site-Specific Biomolecule Labeling with Gold Clusters 241
Abstract 241
1. Introduction 242
2. General Considerations 247
3. Monolayer Protected Cluster Labeling of Biomolecules 249
4. Nanogold Labeling of Proteins 258
5. Ni(II)-NTA-Nanogold Labeling of His-Tagged Proteins 268
6. Conclusions 271
References 271
Chapter 10: How to Operate a Cryo-Electron Microscope 277
Abstract 277
1. Introduction 278
2. Basic Operations of Cryo-EM 279
3. Several Practicals on Imaging Frozen-Hydrated Biological Specimens 289
4. Concluding Remarks 294
References 295
Chapter 11: Collecting Electron Crystallographic Data of Two-Dimensional Protein Crystals 297
Abstract 297
1. Introduction 298
2. Challenges 299
3. Images and Diffraction Patterns 301
4. Specimen Preparation 303
5. Specimen Flatness 310
6. Data Collection 312
Acknowledgments 326
References 326
Chapter 12: Automated Data Collection for Electron Microscopic Tomography 329
Abstract 329
1. Introduction 330
2. Predictive EMT Data Collection 333
3. Real-Time Tomographic Reconstruction 343
4. Dual-Axis EMT Data Collection 349
5. Supporting Automation Procedures 355
6. Summary 358
Acknowledgments 359
References 359
Chapter 13: Correlated Light and Electron Cryo-Microscopy 363
Abstract 363
1. Introduction 363
2. Correlative FLM/ECM with Freezing after FLM Imaging 365
3. Correlative FLM and ECM, with Freezing before FLM Imaging 368
Acknowledgments 386
References 386
Chapter 14: Phase Plates for Transmission Electron Microscopy 389
Abstract 390
1. Introduction 390
2. Types of Phase Plates and Historical Notes 393
3. Microscope Requirements and Modifications 402
4. Phase Plate Operation Procedures 405
5. Summary and Future Prospects 412
Acknowledgments 413
References 413
Chapter 15: Radiation Damage in Electron Cryomicroscopy 417
Abstract 417
1. Introduction 418
2. Measuring Electron Exposure 418
3. Energy Absorbed by Specimens 420
4. Radiation Damage and Choice of Accelerating Voltage 420
5. Primary and Secondary Damage to Proteins During Irradiation 423
6. Tertiary Damage to Proteins During Irradiation 424
7. Cryoprotection and Optimal Temperatures 425
8. Quantification of Beam Damage with Increasing Exposure 427
9. Optimal Exposures for Thin Crystals 429
10. Optimal Exposures for Single Particle Samples 430
11. Optimal Exposures for Tomographic Samples 432
12. Concluding Comments 432
Acknowledgments 433
References 433
Author Index 435
Subject Index 447
Colorplate 457