cryoEM: Methods and Protocols (Methods in Molecular Biology, 2215)

✍ Scribed by Tamir Gonen (editor), Brent L. Nannenga (editor)

• Publisher: Humana
• Year: 2020
• Tongue: English
• Leaves: 346
• Category: Library

✦ Synopsis

This volume details the most up-to-date cryo-EM techniques from leading researchers. Chapters are organized into four parts with emphasis on electron cryotomography, single particle analysis, and the crystal based cryo-EM methods of 2D electron crystallography, and MicroED for the study of 3D crystals. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, CryoEM: Methods and Protocols aims to serve as an excellent resource on cryo-EM and can serve as the foundation for new researchers to this growing field in structural biology.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Electron Cryotomography
Chapter 1: Getting Started with In Situ Cryo-Electron Tomography
1 Introduction
2 Materials
2.1 Growing Cells on Grids
2.1.1 Grid Preparation
Carbon Coating (Optional)
Extracellular Matrix Coating (Optional)
2.1.2 Cell Seeding
2.2 Sample Vitrification
2.3 Data Collection
2.4 Data Processing
3 Methods
3.1 Growing Cells on Grids
3.1.1 Grid Preparation
3.1.2 Cell Seeding
3.2 Sample Vitrification
3.2.1 Preparation of Concentrated Gold Fiducial Marker Solution
3.2.2 Filter Paper and Teflon Sheet Preparation
3.2.3 Plunge Freezing
3.3 Data Collection
3.3.1 Sample Loading and Collection of Whole Grid Montage
3.3.2 Collection of Polygon Maps
3.3.3 Identifying Points of Interest and Tilt Series Collection
3.4 Tomogram Generation
3.5 Segmentation Model Generation
3.5.1 Generate Segmentation Model
3.6 Analysis of Segmentation Model
4 Notes
References
Chapter 2: Cryo-Electron Tomography and Automatic Segmentation of Cultured Hippocampal Neurons
1 Introduction
2 Materials
2.1 Grid Preparation
2.2 PDL Coating
2.3 Neuronal Culture
2.4 Fiducial Gold
2.5 Vitrification
2.6 Cryo-Electron Tomography
2.7 Image Processing Software
3 Methods
3.1 Grid Preparation
3.2 Poly-d-Lysine Coating
3.3 Neuronal Culture
3.4 Preparation of BSA-Coated Gold Fiducials
3.5 Vitrification
3.6 Imaging
3.6.1 Set Imaging Modes
3.6.2 Collect Atlas
3.6.3 Set Eucentric Height
3.6.4 Grid Square Exploration
3.6.5 Search for Target Areas
3.6.6 Select Specific Targets for Data Collection
3.6.7 Saving Tilt-Series Positions
3.6.8 Start Data Collection
3.7 Preprocessing and Reconstruction
3.7.1 Pre-processing
3.7.2 Coarse Alignment
3.7.3 Fiducial Model Generation
3.7.4 Fine Alignment
3.7.5 Tomogram Positioning
3.7.6 Final Aligned Stack
3.7.7 Tomogram Generation
3.7.8 Post-processing
3.8 Automated Segmentation with EMAN2
3.9 Visualization of Segmentation Results
4 Notes
References
Chapter 3: Practical Approaches for Cryo-FIB Milling and Applications for Cellular Cryo-Electron Tomography
1 Introduction
1.1 Principles of Operation
1.1.1 Platinum Sputtering and GIS Deposition
1.1.2 Stage and Shuttle
1.1.3 Stage Temperature Control
1.2 Considerations for Sample Milling Angle and Orientation
2 Materials
2.1 Equipment
2.2 Consumables
3 Methods
3.1 Sample Type Considerations
3.1.1 Mammalian and Flat Eukaryotic Cells
Grid Preparation
Evaluating Grid Quality and Milling
3.1.2 Yeast and Tall Eukaryotic Cells
Grid Preparation
Evaluating Grid Quality and Milling
3.1.3 Bacteria
Grid Preparation
Evaluating Grid Quality and Milling
3.1.4 Cryo-CLEM
3.2 General FIB Milling Protocol
3.2.1 Before Starting
3.2.2 Day of Milling
Microscope Startup and Cooldown
Sample Preparation and Loading
Sample Inspection and Milling Preparation
Lamella Milling
Sample Retrieval
End of Session Tasks
3.3 Considerations for TEM
3.4 Common Troubleshooting Items
3.4.1 System Maintenance
3.4.2 Equipment Troubleshooting
Poor Chamber Vacuum
Frost on Heat Exchanger Tubing
Amorphous Ice Contamination
Crystalline/Atmospheric Ice Contamination
Inaccurate Ion Beam Currents
3.4.3 Sample Troubleshooting
FIB Image Instability
Lamella Appears Skewed Relative to Milling Axis
Curtaining Artifacts
Broken Lamellae at the TEM
Lamella Appears to Bend during Milling
4 Notes
References
Chapter 4: In Situ Imaging and Structure Determination of Biomolecular Complexes Using Electron Cryo-Tomography
1 Introduction
2 Sample Preparation
2.1 Single Small Cells
2.1.1 Bacterial Cultures
2.1.2 Grids and Gold Fiducials
2.1.3 Plunge Freezing of Bacterial Cells
2.2 Eukaryotic Cells
2.2.1 Preparing Cells
2.2.2 Preparing EM Grids for Tissue Culture
2.2.3 Growing Mammalian Cells on Grids
2.2.4 Freezing Grids, Including Adding 20 nm Gold Beads and Fluorescent Microspheres for Cryo-CLEM
2.2.5 Cryo-LM Imaging for Cryo-CLEM Using the FEI Cryostage
2.2.6 FIB-Milling for Imaging Thicker Regions of the Cell
2.3 Biological (Plant) Tissues
2.3.1 Tissue Cultivation
2.3.2 High Pressure Freezing of Plant Tissues
2.3.3 Dissection and Freezing of Plant Tissues
2.3.4 Liquid Cultured Plant Cells
2.3.5 Sample Position Strategy
2.4 Cryosectioning of Plant Tissues
2.4.1 Preparation
2.4.2 Trimming
2.4.3 Trimming Domes from the 200 μm Carrier
2.4.4 Trimming the Type a 100 μm Deep Carrier
2.5 Other Samples
3 Data Collection
3.1 Data Acquisition Using SerialEM: Conventional Tilt-Series
3.2 Rapid Tilt-Series Acquisition
3.2.1 Pre-data Collection Steps
3.2.2 Continuous Tilting Data Collection in SerialEM
3.2.3 Fast-Incremental Data Collection in SerialEM
3.3 Data Collection for High-Resolution (Near-Atomic) Tomography
3.4 Imaging Fluorescent Targets in the Titan Krios Using Cryo-CLEM
4 Data Processing and Tomographic Reconstruction
4.1 Conventional Tilt-Series
4.1.1 Pre-processing
4.1.2 Coarse Alignment
4.1.3 Fiducial Model Generation
4.1.4 Fine Alignment
4.1.5 Tomogram Positioning
4.1.6 Final Aligned Stack
4.1.7 Tomogram Generation
4.2 Fiducial-Less Patch Tracking Using IMOD
4.3 Rapid Tilt-Series Processing
4.3.1 Continuous tilting data
4.3.2 Fast-Incremental Data
4.4 Data Processing for High-Resolution Tomography
4.5 Subtomogram Averaging
5 Notes
References
Part II: Single Particle Analysis and Electron Cryomicroscopy
Chapter 5: Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples
1 Introduction
2 Materials
2.1 Grid Handling and Preparation
2.2 Stain Solution: Preparation and Storage
2.3 Staining Workflow
3 Methods
3.1 Uranyl Acetate (2%) (Concentration Can be Adjusted)
3.2 Uranyl Formate (see Note 4)
3.3 Staining Protocol
4 Notes
References
Chapter 6: Setting Up Parallel Illumination on the Talos Arctica for High-Resolution Data Collection
1 Introduction
2 Materials
2.1 Equipment
2.2 Software
3 Methods
3.1 Basic Column Alignments
3.2 Coma-Free Alignment
3.3 Parallel Illumination
3.4 Determine and Set the Exposure Rate
3.5 K2 Gain Corrections
4 Notes
References
Chapter 7: Multi-body Refinement of Cryo-EM Images in RELION
1 Introduction
2 Materials
2.1 Computer Hardware Requirements
2.2 Software Requirements
2.3 Test Data
3 Methods
3.1 Prepare Input Files
3.2 Execute Multi-body Refinement
3.3 Analyze the Body Reconstructions
3.4 Analyze the Body Motions
3.5 Partial Signal Subtraction after Multi-body Refinement
4 Notes
References
Chapter 8: Evaluating Local and Directional Resolution of Cryo-EM Density Maps
1 Introduction
2 Materials
2.1 Software
2.2 Cryo-EM Maps
3 Methods
3.1 Estimating and Evaluating the Local Resolution of a Reconstructed Map
3.2 Estimating and Evaluating the Directional Resolution of a Reconstructed Map
3.3 Analyses of the Individual Results
3.3.1 Half-Map and Map-Model FSCs
3.3.2 Interpreting the Local Resolution of the Map
3.3.3 Interpreting the Shape of the 3D FSC Volume
3.3.4 Interpreting the Experimental Map
4 Notes
References
Chapter 9: Automated Modeling and Validation of Protein Complexes in Cryo-EM Maps
1 Introduction
2 Fit Assessment
2.1 Comparing Structure and Maps
2.2 Map Blurring
2.3 Scoring Functions
2.3.1 Global Scoring
Cross-Correlation
Mutual Information
Overlap Score
2.3.2 Local Scoring
Local Cross-Correlation: SCCC
SMOC
2.3.3 Consensus Scoring
3 Structure-Density Fitting
3.1 Protein Structure Prediction
3.2 Rigid-Body Fitting
3.3 Assembly Fitting
3.3.1 Initial Placement
3.3.2 IQP and wICP
3.3.3 γ-TEMPy (Genetic Algorithm for Modeling Macromolecular Assemblies with Template and EM Comparison Using Python)
3.4 Flexible Fitting (Refinement)
3.4.1 Flex-EM
3.4.2 RIBFIND
3.4.3 Resolution-Dependent Refinement
3.5 Consensus Refinement Approach
3.6 Hierarchical Refinement Approach
3.6.1 Loop Optimization
3.6.2 Ligand Fitting
3.7 Model Validation
3.7.1 Assessing Global Quality
Map Assessment
Structure Assessment
Model Validation by Additional Experiments
Jwalk and MNXL: Model validation Using Cross-Linking Mass Spectrometry
3.7.2 Assessing the Local Quality
Map Assessment
Local Structure
4 Summary
5 Methods
5.1 Candidate Model Generation
5.2 Rigid-Body Fitting
5.2.1 Finding Rigid Bodies with RIBFIND
5.3 TEMPy
5.3.1 Structure Blurring
5.3.2 Scoring Fits
5.3.3 Ensemble Generation and Scoring
5.4 γ-TEMPy
5.5 Flexible Fitting with Flex-EM
5.6 Density Difference Mapping
5.7 Fitting Small Molecules
5.8 Jwalk and MNXL
5.9 CCP-EM
6 Notes
6.1 Resolution
6.2 Density Map Preprocessing
6.3 Cross-Correlation and Manders´ Coefficients
6.4 Model Generation
6.5 Rigid-Body Fitting
6.6 RIBFIND
6.7 Flex-EM
6.8 Difference Maps
6.9 Docking
6.10 Jwalk
References
Part III: Electron Crystallography of 2D Crystals
Chapter 10: 2D Electron Crystallography of Membrane Protein Single-, Double-, and Multi-Layered Ordered Arrays
1 Introduction
2 Materials
2.1 Two-Dimensional Crystallization Trials
2.2 Negative Staining of 2D Crystallization Trials
2.3 TEM Screening of Crystallization Trials
2.4 Cryo-EM Grid Preparation, Screening and Data Collection
2.5 Image Processing
3 Methods
3.1 Crystallization of Membrane Proteins Through Detergent Removal by Dialysis
3.1.1 Preparation of Solubilized Lipid Stock
3.1.2 Setup of 2D Crystallization Trials
3.2 Negative Stain TEM Screening for 2D Crystallization Conditions
3.2.1 Negative Staining
3.2.2 Screening by TEM
3.2.3 Refinement of Crystallization Conditions
3.2.4 Decision on Cryo-EM Approach
3.3 Cryo-EM Grid Preparation and Cryo-EM
3.3.1 Cryo-EM Grid Preparation of Single- Or Double-Layered 2D Crystals
3.3.2 Peel-Blot
3.3.3 Initial Assessment of Cryo-EM Conditions
3.3.4 Cryo-EM Data Collection
3.4 Image Processing
4 Notes
References
Chapter 11: Sample Preparation and Data Collection for Electron Crystallographic Studies on Membrane Protein Structures and Li...
1 Introduction
2 Materials
2.1 Grid Specimen Preparation
2.2 Frozen Grid Specimen Transfer
3 Methods
3.1 Sample Preparation
3.1.1 Preparation of the Thin Carbon Films in Spark-Free Conditions
3.1.2 Back-Injection Method
3.1.3 Carbon-Sandwich Method
3.2 Data Collection
3.2.1 Electron Microscope Checkup
3.2.2 Beam Alignment
3.2.3 Electron Diffraction
3.2.4 Electron Imaging
3.3 Initial Data Quality Assessment
4 Notes
References
Chapter 12: Single Particle Analysis for High-Resolution 2D Electron Crystallography
1 Introduction
2 Materials
2.1 Software and Hardware
3 Methods
3.1 Data Acquisition and Initial Processing of Movies
3.2 Conventional 2D Crystallographic Reconstruction
3.3 Exporting a 2D crystal Project for Single Particle Analysis
3.3.1 Particle Picking
3.3.2 CTF Correction
3.3.3 Exporting the Metadata
3.4 Pre-Refinement
3.4.1 Crystal Averages
3.4.2 Running the Pre-Refinement
3.5 Consensus Refinement
3.6 3D Classification
3.7 Assessing Resolution and Post-Processing
3.8 Conclusions
4 Notes
References
Part IV: Microcrystal Electron Diffraction (MicroED)
Chapter 13: MicroED Sample Preparation and Data Collection For Protein Crystals
1 Introduction
2 Materials
3 Methods
3.1 Sample Preparation
3.2 Screening Microcrystal Diffraction Quality
3.3 MicroED Data Collection
4 Notes
References
Chapter 14: Detection of Microcrystals for CryoEM
1 Introduction
2 Materials
2.1 Microscopy
2.2 Negative Staining Components
3 Methods
4 Notes
References
Chapter 15: Low-Dose Data Collection and Radiation Damage in MicroED
1 Introduction
2 Sample Preparation
3 Data Collection
3.1 Cryo-EM
3.2 Resolution Effects
3.3 Exposure and Dose
3.4 Multicrystal Data Collection
4 Camera Considerations
5 Conclusion and Outlook
References
Chapter 16: Automation of Continuous-Rotation Data Collection for MicroED
1 Introduction
2 Materials
2.1 Microscope
2.2 Electron Detector
2.3 Image Acquisition Software
3 Methods
3.1 Microscope and Software Setup
3.2 Crystal Screening
3.3 Testing Crystals for Diffraction in Batch
3.4 Preparing Crystals for Data Collection (See Note 2)
3.5 Data Acquisition (See Note 3)
4 Notes
References
Chapter 17: Ab Initio Determination of Peptide Structures by MicroED
1 Introduction
2 Materials
2.1 Sample-Preparation Tools and Consumables
2.2 Grid Surface Treatment
2.3 Cryo Storage and Cryo-Transfer Systems
2.4 Microscope Components
2.5 Cameras and Computing
2.6 Data Reduction and Structure Determination Software
3 Methods
3.1 Grid Preparation and Freezing (See Note 4)
3.2 Cryo-Transfer (See Note 4)
3.3 Grid Screening and Preliminary Diffraction
3.4 Data Conversion and Processing
3.5 Dataset Merging
3.6 Structure Determination
4 Notes
References
Index