The Resolution Revolution: Recent Advances In cryoEM

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cryoEM, a new volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods and new developments in recording images, the creation, evaluation and validation of 3D maps from the images, model building into maps and refinement of the resulting atomic structures, and applications of essentially single particle methods to helical structures and to sub-tomogram averaging.

Continues the legacy of this premier serial with quality chapters authored by leaders in the field
Covers research methods that determine the structures of biological molecules, a vital step for understanding their function
Contains the technical developments underpinning the advances of cryoEM and captures the exciting insights that have resulted

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Front Cover 2
The Resolution Revolution: Recent Advances In cryoEM 5
Copyright 6
Contents 7
Contributors 13
Preface 15
- References 20
Chapter One: Direct Electron Detectors 23
- 1. Introduction 23
- 2. Past 24
- 3. Present 26
  - 3.1. Practical Advice for the User 32
- 4. Future 35
- References 37
Chapter Two: Specimen Behavior in the Electron Beam 41

Front Cover 2
The Resolution Revolution: Recent Advances In cryoEM 5
Copyright 6
Contents 7
Contributors 13
Preface 15
- References 20
Chapter One: Direct Electron Detectors 23
- 1. Introduction 23
- 2. Past 24
- 3. Present 26
  - 3.1. Practical Advice for the User 32
- 4. Future 35
- References 37
Chapter Two: Specimen Behavior in the Electron Beam 41
- 1. Introduction 42
- 2. High-Energy Electrons Are a Form of Ionizing Radiation as Well as Being a Form of Short-Wavelength Radiation That Can B ... 43
  - 2.1. Electron-Scattering Events Can Be Either Elastic or Inelastic 43
  - 2.2. Energy Is Deposited in the Specimen as a Result of Inelastic Scattering 44
  - 2.3. Values of the Linear Energy Transfer (LET) Can Be Used to Estimate the Energy Deposited 46
- 3. Biological Molecules Become Structurally Damaged When Irradiated 47
  - 3.1. There Is a Large Literature of Radiation Chemistry and Radiation Biology 47
  - 3.2. Fading of Diffraction Patterns Is a Convenient Indicator of Structural Damage 49
  - 3.3. Some Residues in Proteins Are Especially Sensitive to Radiation Damage 50
  - 3.4. Caging of Fragments and ``Trapping´´ of Radicals Results in Cryo-Protection: This Helps Only to a Limited Extent 51
  - 3.5. Radiation Sensitivity of Enzyme Activity: Implications for Dynamic Studies in Liquid Samples 52
- 4. Vitreous Ice Also Becomes Structurally Damaged by Ionizing Radiation 52
  - 4.1. Water Molecules Are Easily Damaged by Ionizing Radiation 52
  - 4.2. Weak Thon Rings at High Resolution Show That Vitreous Ice Is Very Sensitive to Radiation Damage 53
  - 4.3. Electron-Stimulated Desorption Progressively Thins Ice Specimens 55
- 5. Bubbling of Hydrated Biological Specimens Becomes Apparent at High Electron Exposure 56
  - 5.1. Bubbles Consist of Molecular Hydrogen 57
  - 5.2. Bubbling Can Be Used to Evaluate the Specimen Thickness 58
  - 5.3. Bubbling Can Be Used to Distinguish Regions with Different Chemical Composition (Bubblegrams) 58
- 6. Cryo-Specimens Exhibit Collective Beam-Induced Movement When Irradiated 59
  - 6.1. Radiation-Sensitive Specimens Show Beam-Induced Motion at Quite Low Electron Exposures 59
  - 6.2. Thin Cryo-Specimens Undergo Drum-Head-Like Flexing and Doming When Irradiated 60
  - 6.3. Images Can Be Corrupted Significantly by There Being Changes in Z-Height 61
  - 6.4. The Pattern of Beam-Induced Movement Can Be Quite Unpredictable 61
- 7. More Than One Mechanism May Contribute to Beam-Induced Motion 62
  - 7.1. Cryo-EM Specimens, as Made, Are Expected to Be Under Considerable Stress 62
  - 7.3. Irradiation Can Generate (New) Mechanical Stress 63
  - 7.2. Irradiation Can Relieve Mechanical Stress 63
  - 7.4. Which Comes First, Relaxation or Creation of Stress? 64
- 8. Irradiation Can Produce Electrostatic Charging of the Specimen 64
  - 8.1. A Buildup of Net Charge on the Specimen Can Be Easy to Detect 65
  - 8.2. Evidence of Net-Charge Buildup Can Be Reduced in Several Ways 65
  - 8.3. Other Forms of Specimen Charging Are More Subtle to Detect 66
- 9. Summary and Future Directions 68
- References 69
- Acknowledgments 69
Chapter Three: Specimen Preparation for High-Resolution Cryo-EM 73
- 1. Introduction 74
- 2. A Systematic Approach to Specimen Preparation 75
  - 2.1. Protein Preparation 77
  - 2.3. Diagnostic Cryo-EM 78
  - 2.2. Negative Stain 78
  - 2.4. Initial Cryo-EM Data Collection 81
  - 2.5. High-Resolution Data Collection 82
- 3. Support Choice, Handling, and Storage 84
- 4. Contamination and Cleaning 87
- 5. Continuous Films of Amorphous Carbon and Graphene 89
- 6. Surface Treatments 95
- 7. Vitrification 98
- 8. Data Collection 102
- References 106
- Acknowledgments 106
Chapter Four: Strategies for Automated CryoEM Data Collection Using Direct Detectors 109
- 1. Introduction 110
- 2. Practical Considerations in Instrumentation Configuration 112
- 3. Automated Data Acquisition Using DD Cameras 115
  - 3.1. Gain/Dark Reference Image Preparation 115
  - 3.2. Choosing Dose Rates 116
  - 3.3. Choosing Total Exposure Time and Dose 116
  - 3.4. Frame Saving and Drift Correction 117
- 4. Challenges Arising from DDs and High-Resolution Biological CryoEM 117
  - 4.1. High-Resolution Data Collection Is Slower 117
  - 4.2. Computing Requirements 120
- 5. Future Outlook for DD Integration with Automated Data Collection 122
- References 123
- Acknowledgments 123
Chapter Five: Processing of Cryo-EM Movie Data 125
- 1. Introduction 125
- 2. Motioncorr 129
- 3. alignframes_lmbfgs and alignparts_lmbfgs 131
- 4. Unblur 134
- 5. Optical Flow 136
- 6. Particle Polishing in Relion 138
- 7. Summary 143
- 8. Future Prospects 144
- Acknowledgments 145
- References 145
Chapter Six: Processing of Structurally Heterogeneous Cryo-EM Data in RELION 147
- 1. Introduction 148
- 2. New Algorithmic Concepts 150
  - 2.1. Regularization: The Empirical Bayesian Approach 150
  - 2.2. Prevention of Overfitting: The Gold-Standard Approach to Refinement 151
  - 2.3. Getting Clean, High-Resolution Maps: The Postprocessing Approach 153
  - 2.4. Beam-Induced Motion Correction and Radiation-Damage Weighting: The Movie-Processing Approach 154
- 3. A Typical High-Resolution Structure Determination Procedure 156
- 4. Dealing with Structural Heterogeneity 164
  - 4.1. 3D Classification with Exhaustive Angular Searches 164
  - 4.2. Detection of Remaining Structural Heterogeneity 165
  - 4.4. Masked 3D Auto-Refinement 166
  - 4.3. 3D Classification with Finer, Local Angular Searches 166
  - 4.5. Masked 3D Classification 168
  - 4.6. Masked 3D Refinement/Classification with Partial Signal Subtraction 169
  - 4.7. Dealing with Pseudo-Symmetry 172
  - 4.8. Multibody Refinement 173
  - 4.9. A More Elaborate Example 174
- References 176
- 5. Outlook 176
- Acknowledgments 176
Chapter Seven: Single-Particle Refinement and Variability Analysis in EMAN2.1 181
- 1. Introduction 182
  - 1.1. History 182
  - 1.2. The Scope of the Problem 183
  - 1.3. EMAN2.1 Philosophy 184
- 2. Single-Particle Reconstruction 186
- 3. Assessing a Refinement and Identifying Variability 189
  - 3.1. Resolution 189
  - 3.2. Map Accuracy, Variability, and Symmetry 190
  - 3.3. Model-Based Refinement 193
- 4. Conformational and Compositional Variability 194
  - 4.1. Variance Analysis 194
  - 4.2. Multimodel Refinement 195
  - 4.3. Splitting 3-D Refinements Using 2-D Principal Component Analysis 197
  - 4.4. Model-Based Binary Separation in 3-D 198
  - 4.5. Robust Extraction of Particle Fragments 200
  - 4.6. Breaking Symmetry 202
  - 4.7. Subtomogram Averaging 203
  - 4.8. Tilt Validation 205
- 5. Interactive Tools 206
- 6. Conclusion 208
- Acknowledgments 209
- References 209
Chapter Eight: Frealign: An Exploratory Tool for Single-Particle Cryo-EM 213
- 1. Introduction and Philosophy 214
- 2. Frealign Elements at a Glance 215
  - 2.1. Running Frealign 215
  - 2.2. Required Input 216
  - 2.3. Optional Input 219
  - 2.4. Output 219
  - 2.5. Naming Conventions 220
- 3. Algorithms 221
- 4. Typical Application Scenarios 223
  - 4.1. Refinement of a Structure Generated with Different Software 223
  - 4.2. 3D Reconstruction Using Parameters from a Previous Frealign Run 230
  - 4.3. 3D Classification 230
  - 4.4. Selecting or Merging Particles from Different Classes 232
  - 4.5. Generating an Initial Map 233
  - 4.6. Using Masks 235
  - 4.7. Asymmetric Refinement 238
  - 4.8. Processing Images of Helical Structures 241
- 5. Tuning Options 242
- 6. Related Software 245
- References 246
Chapter Nine: Testing the Validity of Single-Particle Maps at Low and High Resolution 249
- 1. Introduction 250
- 2. Validation at Low Resolution: Tilt-Pair Analysis and Orientation Determination 252
  - 2.1. The Tilt-Pair Experiment 254
  - 2.2. Tilt-Pair Parameter Plot 255
  - 2.3. Tilt-Pair Phase Residual Plot 256
  - 2.4. Map Validation 258
  - 2.5. Hand Discrimination 259
  - 2.6. Optimization of Orientation Determination and Alignment Accuracy 260
  - 2.7. Alignment Reliability Test 261
- 3. Validation of Map Resolution 261
  - 3.1. The High-Resolution Noise-Substitution Test 264
  - 3.2. The Effect of Masks 266
  - 3.3. Another Noise Test 267
- 4. Resolution Assessment of a Map with an Atomic Coordinate Model 267
- 5. Contrast Restoration 268
- 6. Local Resolution Assessment 269
- 7. Summary of Validation Tests and Future Prospects 271
- References 272
- Acknowledgments 272
Chapter Ten: Tools for Model Building and Optimization into Near-Atomic Resolution Electron Cryo-Microscopy Density Maps 277
- 1. Introduction 277
- 2. De Novo Model Building 280
- 3. Model Optimization 284
- 4. Model Validation 291
- 5. Discussion 295
- References 296
- Acknowledgments 296
Chapter Eleven: Refinement of Atomic Structures Against cryo-EM Maps 299
- 1. Introduction 300
  - 1.1. Notation 302
- 2. Target Function 303
  - 2.1. Prior Knowledge 305
  - 2.2. Likelihood 306
- 3. Variance of Observational Noise 308
  - 3.1. Half-Data Reconstruction 309
  - 3.2. Noise Reconstruction 309
- 4. Map Calculation 310
- 5. Crossvalidation 312
- 6. Effect of Oversharpening 314
- 7. 2Fo-Fc and Other Crystallographic Maps 316
- 8. Form Factors 317
- 9. Conclusions 321
- Acknowledgments 324
- References 324
Chapter Twelve: Cryo-EM Structure Determination Using Segmented Helical Image Reconstruction 329
- 1. Introduction 330
- 2. Architecture of Helical Assemblies 332
- 3. Segmented Helical Image Analysis 333
- 4. Iterative Helical Structure Refinement 337
- 5. High-Resolution Helical Image Reconstructions 339
- 6. The Use of Direct Electron Detectors for Helical Structures 341
- 7. Validation of Helical Structures 344
- 8. Conclusion 345
- Acknowledgments 346
- References 347
Chapter Thirteen: Cryo-Electron Tomography and Subtomogram Averaging 351
- 1. Introduction 352
- 2. Tomographic Data Collection 355
  - 2.1. General Principles 355
  - 2.2. Tomogram Acquisition 356
- 3. Tilt-Series Processing and Tomogram Reconstruction 359
  - 3.1. Preprocessing of Raw Images 359
  - 3.2. Defocus Determination of Tilt Images 360
  - 3.3. CTF Correction of Tilt Images 362
  - 3.4. Local Defocus Determination and CTF Correction 363
  - 3.5. Tilt-Series Alignment 364
  - 3.6. Tomogram Reconstruction 365
- 4. Subtomogram Averaging 367
  - 4.1. Particle Picking and Starting References 367
  - 4.2. General Principles of Subtomogram Averaging 368
  - 4.3. Alignment by Angular Search 370
  - 4.4. Alignment with Fast Rotational Matching 372
  - 4.5. Postprocessing: Assessing and Reweighting the Final Structure 373
- 5. Classification in Subtomogram Averaging 373
  - 5.1. General Principles 373
  - 5.2. Postalignment Classification 374
  - 5.3. Classification During Alignment 376
- 6. Maximum-Likelihood Approaches for Subtomogram Alignment and Classification 377
  - 6.1. General Principles 377
  - 6.2. Current ML Implementations 378
- 7. Combinations of Subtomogram Averaging and 2D Approaches 379
  - 7.1. Using 3D Data to Aid 2D Reconstructions 379
  - 7.2. Improving Subtomogram Averages with 2D Alignment 380
- 8. Software for Subtomogram Averaging 382
- 9. Conclusions 383
- Acknowledgments 384
- References 384
Chapter Fourteen: High-Resolution Macromolecular Structure Determination by MicroED, a cryo-EM Method 391
- 1. Introduction 392
- 2. Background 393
  - 2.1. Origins of Electron Diffraction 393
  - 2.2. Transmission Electron Microscopy and Diffraction 393
  - 2.3. Electron Diffraction of Protein Assemblies 394
  - 2.4. Electron Diffraction of 3D Protein Crystals: MicroED 395
- 3. Sample Preparation 400
  - 3.1. Approaches to Growth and Screening of Crystals for MicroED 400
  - 3.2. Cryo-Preservation of Nanocrystals for MicroED 401
- 4. Instrumentation and Data Collection 402
  - 4.1. Electron Source Specifications 402
  - 4.2. Electron Optics Specifications 402
  - 4.3. Energy Filtering 404
  - 4.4. Low-Dose Data Collection 405
  - 4.5. Detector Specifications 406
- 5. Processing of MicroED Data 406
  - 5.1. Conversion and Processing of Diffraction Images 406
  - 5.2. Structure Determination and Refinement 409
- 6. Summary and Outlook 410
- Acknowledgments 410
- References 411
Chapter Fifteen: Databases and Archiving for CryoEM 415
- 1. Introduction 416
- 2. Resources 421
  - 2.1. Searching, Browsing, and Visualizing Data 421
  - 2.2. Validation 422
    - 2.2.1. Visual Analysis Pages 422
    - 2.2.2. Stand-Alone Validation Servers 422
    - 2.2.3. Validation Pipeline 423
  - 2.3. Programmatic Access 424
- 3. Deposition and Annotation 424
  - 3.1. EMDB and PDB 424
  - 3.2. EMPIAR 425
- 4. Recent Community-Wide Initiatives 425
  - 4.1. EM Validation Task Force 425
  - 4.2. ``Data Management Challenges in 3D Electron Microscopy´´ Workshop 426
  - 4.3. ``A 3D Cellular Context for the Macromolecular World´´ Workshop 427
  - 4.4. wwPDB Hybrid/Integrative Methods Task Force Workshop 428
  - 4.5. EMDataBank Map and Model Validation Challenges 429
- 5. Challenges and Opportunities 430
  - 5.1. Rise of Multiuser Facilities, CCP-EM, and Prospects for Data Harvesting 430
  - 5.2. New Imaging Modalities 431
- Acknowledgments 431
- References 432
Author Index 435
Subject Index 453
Color Plate 469
Back Cover 489

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