Cryo-EM Part A: Sample Preparation and Data Collection

Series Editors
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Copyright
......Page 2
Contributors
......Page 3
Preface
......Page 7
Volume in Series
......Page 9
3D Reconstruction from Electron Micrographs: A Personal Account of its Development
......Page 38
Viruses......Page 39
Helical Structures......Page 44
3D Reconstruction of Helical Structures......Page 47
Digital Image Processing......Page 49
3D Reconstruction of Asymmetric Structures......Page 54
References......Page 61
Preparation of 2D Crystals of Membrane Proteins for High-Resolution Electron Crystallography Data Collection......Page 62
Purification of Membrane Proteins......Page 63
2D Crystallization of Membrane Proteins......Page 65
Methods......Page 67
Requirements for Electron Crystallography Data Collection......Page 69
Electron microscopy grid preparation for 2D crystals......Page 70
Preparation of flat support films......Page 71
Back injection method......Page 72
2D crystal grid preparation with the carbon sandwich method......Page 73
Reducing beam-induced resolution loss on tilted specimens......Page 74
Conclusions......Page 77
References......Page 78
Helical Crystallization of Soluble and Membrane Binding Proteins......Page 81
Streptavidin and perfringolysin O: Model proteins for helical crystallization on lipid nanotubes......Page 82
Liposomes as substrates for helical crystallization......Page 83
Dynamin and botulinum toxin as prototypes for liposome-mediated helical crystallization......Page 84
Lipids......Page 85
Helical crystallization of streptavidin......Page 86
Nanotube preparation for perfringolysin O......Page 87
Helical crystallization of wild-type and mutant PFO......Page 88
Helical crystallization of wild-type PFO for 3D map......Page 89
Helical crystallization of dynamin......Page 90
Helical crystallization of BoNT......Page 92
Alternative strategies and future outlooks......Page 93
References......Page 94
Plunge Freezing for Electron Cryomicroscopy......Page 99
Introduction......Page 100
Grids and Supports......Page 101
Cleaning the Grids......Page 102
Preparing the Cryogen......Page 104
Condensing the cryogen......Page 105
Safety considerations......Page 106
Basic procedure......Page 107
Controlling humidity......Page 109
Instrumentation......Page 110
Common Problems and Their Diagnoses......Page 113
Acknowledgments......Page 114
References......Page 116
A Practical Guide to the Use of Monolayer Purification and Affinity Grids......Page 119
A Brief History of the Use of Lipid Monolayers in Electron Microscopy......Page 121
Preparation of Lipid Monolayer Specimens......Page 122
Sample level......Page 124
Protocol for monolayer purification......Page 125
Advantages of monolayer purification......Page 126
Limitations of monolayer purification......Page 127
Ribosomal complexes-Purifying complexes from cell extract......Page 128
C complex-Preparing labile complexes that usually cannot be vitrified......Page 129
The Affinity Grid......Page 130
Use of Affinity Grids for complexes without a His tag......Page 131
Antibody concentration and incubation time......Page 132
Advantages of the Affinity Grid......Page 133
Examples of Affinity Grid Applications......Page 134
The complex of Tf-TfR with GP1-Recruiting targets using tagged ligands......Page 135
Aquaporin-9-Purification of membrane proteins......Page 136
The Notch extracellular domain-Purification of proteins with low expression levels......Page 137
Characterization of Monolayer Specimens......Page 138
STEM analysis......Page 139
Challenges and Future Directions......Page 140
References......Page 141
GraFix: Stabilization of Fragile Macromolecular Complexes for Single Particle Cryo-EM......Page 144
Introduction......Page 145
Overview of the GraFix Procedure......Page 147
Promotion of intramolecular cross-linking......Page 148
Weak cross-linking with no apparent structural artifacts......Page 149
Reduction of sample heterogeneity......Page 150
Increased particle binding......Page 152
Coanalysis of proteins within the cross-linked particles......Page 154
Preparing a continuous density gradient......Page 155
Adding a buffering cushion......Page 156
Loading the sample......Page 157
Placing carbon film onto samples......Page 158
Adsorption times......Page 159
References......Page 160
Cryonegative Staining of Macromolecular Assemblies......Page 162
Introduction......Page 163
Cryonegative Staining-The "Adrian" Method: Frozen-Hydrated Specimens in the Presence of a Saturated Ammonium... 
......Page 168
Results obtained with the Adrian method (saturated ammonium molybdate)......Page 170
Advantages and limitations......Page 173
Cryonegative Staining with the Sandwich Method: The "Holger Stark" Alternative
......Page 174
Conclusion......Page 176
References......Page 178
Liposomes on a Streptavidin Crystal......Page 181
Introduction......Page 182
Streptavidin crystals......Page 183
Growth of the streptavidin crystal......Page 184
Crystal transfer and liposome tethering......Page 186
Analysis and removal of crystal information from micrographs......Page 189
Proteoliposomes......Page 190
Purification and reconstitution of BK channels......Page 191
Flux assay to test the function of reconstituted proteins......Page 193
Computational removal of the lipid membrane contribution......Page 194
Using the liposome image to aid the orientation determination of each particle......Page 195
Conclusion......Page 196
References......Page 197
Micromanipulator-Assisted Vitreous Cryosectioning and Sample Preparation by High-Pressure Freezing......Page 199
Introduction......Page 200
Why is Vitreous Cryosectioning so Difficult?......Page 201
High-Pressure Freezing for Vitreous Cryosectioning......Page 202
Extracellular Cryoprotectants for Vitreous Cryosectioning......Page 204
Mounting a Vitrified Sample in the Cryo-Ultramicrotome......Page 205
Hardware: Cryo-Ultramicrotomes......Page 207
The Micromanipulator......Page 210
Cryodiamond Knives......Page 211
Setting up the Cryo-Ultramicrotome for Sectioning......Page 213
Trimming a Blockface......Page 214
Preparing to Cryosection......Page 216
The Active Static Ionizer......Page 217
Cryosectioning......Page 218
Transferring Cryosections to the EM Grid......Page 220
Pressing the Sections onto the Grid......Page 221
The Sectioning Environment and Relative Humidity......Page 223
Cryosectioning Artifacts......Page 225
Conclusion......Page 226
References......Page 227
Site-Specific Biomolecule Labeling with Gold Clusters......Page 229
Introduction......Page 230
General Considerations......Page 235
Stoichiometric labeling......Page 236
Monolayer Protected Cluster Labeling of Biomolecules......Page 237
Design considerations......Page 239
Synthesis of Au144(pMBA)60......Page 241
Assay of Au144(pMBA)60 synthesis......Page 243
Separation of Au144(pMBA)60 conjugates......Page 244
Conjugation of the Nanogold label......Page 246
Separation of Nanogold conjugates......Page 249
Assay of labeling and activity......Page 251
Electron cryomicroscopy with incorporated Nanogold conjugates......Page 253
Ni(II)-NTA-Nanogold Labeling of His-Tagged Proteins......Page 256
References......Page 259
How to Operate a Cryo-Electron Microscope......Page 265
Introduction......Page 266
Microscope startup and cryo-EM grid insertion procedure......Page 267
Basic microscope alignment......Page 269
Low dose mode setup......Page 271
Target search and picture recording......Page 273
Cryo-grid replacement during an EM session......Page 275
Microscope shut down procedure......Page 276
How to quickly estimate the electron dose at the exposure time used and at a particular MAG......Page 277
How to quickly estimate the thickness of vitreous ice......Page 278
How to minimize exposure to the specimen before taking a picture......Page 279
How to minimize the specimen drift and vibration during exposure......Page 280
How to minimize the beam-induced specimen charging and movement during exposure......Page 281
Concluding Remarks......Page 282
References......Page 283
Collecting Electron Crystallographic Data of Two-Dimensional Protein Crystals......Page 284
Introduction......Page 285
Challenges......Page 286
Images and Diffraction Patterns......Page 288
Specimen Preparation......Page 290
Sugar embedding......Page 291
Protocol for room temperature glucose embedding......Page 294
Protocol for trehalose carbon sandwich embedding......Page 295
Vitrification......Page 296
Specimen Flatness......Page 297
Protocol for spark-free preparation of thin carbon films......Page 298
Data Collection......Page 299
Protocol for alignment of the condenser aperture......Page 301
Procedure for aligning the low-dose modes for imaging......Page 303
Collecting images......Page 304
Procedure for collecting images......Page 305
Recording electron diffraction patterns......Page 307
Procedure for recording diffraction patterns......Page 308
Collecting data of tilted specimens......Page 311
References......Page 313
Automated Data Collection for Electron Microscopic Tomography......Page 316
Introduction......Page 317
Geometric model......Page 320
Dynamic determination of z0......Page 322
Experimental verification......Page 323
System implementation......Page 328
General strategy......Page 330
Parallel real-time reconstruction......Page 331
System design and implementation......Page 332
Experimental verification......Page 333
Finding targets for sequential EMT data collection......Page 336
Sequential EMT data collection......Page 338
Rotational alignment......Page 339
Experimental verification......Page 341
Autofocus procedure......Page 342
Autoeucentricity procedure......Page 344
Summary......Page 345
References......Page 346
Introduction......Page 349
Correlative FLM/ECM with Freezing after FLM Imaging......Page 351
Correlative FLM and ECM, with Freezing before FLM Imaging......Page 354
References......Page 372
Phase Plates for Transmission Electron Microscopy......Page 374
Introduction......Page 375
Thin film phase plates......Page 378
Electrostatic phase plates......Page 381
Magnetic phase plates......Page 382
Photonic phase plates......Page 383
Electrostatic mirror pixel-wise phase shifter......Page 385
Anamorphotic phase plates......Page 386
Phase plate holder......Page 387
Illumination system......Page 388
Transfer lenses and aberration correctors......Page 389
Phase Plate Operation Procedures......Page 390
Setting the phase plate on a diffraction plane......Page 391
Phase plate centering......Page 392
Adjusting electron dose......Page 393
Focusing and phase plate condition evaluation......Page 394
Low dose observation with a phase plate......Page 395
Summary and Future Prospects......Page 397
References......Page 398
Radiation Damage in Electron Cryomicroscopy......Page 401
Measuring Electron Exposure......Page 402
Radiation Damage and Choice of Accelerating Voltage......Page 404
Primary and Secondary Damage to Proteins During Irradiation......Page 407
Tertiary Damage to Proteins During Irradiation......Page 408
Cryoprotection and Optimal Temperatures......Page 409
Quantification of Beam Damage with Increasing Exposure......Page 411
Optimal Exposures for Thin Crystals......Page 413
Optimal Exposures for Single Particle Samples......Page 414
Concluding Comments......Page 416
References......Page 417
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