Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ix
PART I ELECTRON CRYOTOMOGRAPHY
1 Getting Started with In Situ Cryo-Electron Tomography . . . . . . . . . . . . . . . . . . . .
3
Daniel Serwas and Karen M. Davies
2 Cryo-Electron Tomography and Automatic Segmentation
of Cultured Hippocampal Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Ryan K. Hylton, Victoria H. Seader, and Matthew T. Swulius
3 Practical Approaches for Cryo-FIB Milling and Applications
for Cellular Cryo-Electron Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Vinson Lam and Elizabeth Villa
4 In Situ Imaging and Structure Determination of Biomolecular Complexes
Using Electron Cryo-Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Mohammed Kaplan, William J. Nicolas, Wei Zhao,
Stephen D. Carter, Lauren Ann Metskas, Georges Chreifi,
Debnath Ghosal, and Grant J. Jensen
PART II SINGLE PARTICLE ANALYSIS AND ELECTRON CRYOMICROSCOPY
5 Progress Towards CryoEM: Negative-Stain Procedures
for Biological Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Shane Gonen
6 Setting Up Parallel Illumination on the Talos Arctica for High-Resolution
Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Mark A. Herzik Jr
7 Multi-body Refinement of Cryo-EM Images in RELION. . . . . . . . . . . . . . . . . . . . 145
Takanori Nakane and Sjors H. W. Scheres
8 Evaluating Local and Directional Resolution
of Cryo-EM Density Maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Sriram Aiyer, Cheng Zhang, Philp R. Baldwin,
and Dmitry Lyumkis
9 Automated Modeling and Validation of Protein Complexes
in Cryo-EM Maps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Tristan Cragnolini, Aaron Sweeney, and Maya Topf
vii
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ix
PART I ELECTRON CRYOTOMOGRAPHY
1 Getting Started with In Situ Cryo-Electron Tomography . . . . . . . . . . . . . . . . . . . .
3
Daniel Serwas and Karen M. Davies
2 Cryo-Electron Tomography and Automatic Segmentation
of Cultured Hippocampal Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Ryan K. Hylton, Victoria H. Seader, and Matthew T. Swulius
3 Practical Approaches for Cryo-FIB Milling and Applications
for Cellular Cryo-Electron Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Vinson Lam and Elizabeth Villa
4 In Situ Imaging and Structure Determination of Biomolecular Complexes
Using Electron Cryo-Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Mohammed Kaplan, William J. Nicolas, Wei Zhao,
Stephen D. Carter, Lauren Ann Metskas, Georges Chreifi,
Debnath Ghosal, and Grant J. Jensen
PART II SINGLE PARTICLE ANALYSIS AND ELECTRON CRYOMICROSCOPY
5 Progress Towards CryoEM: Negative-Stain Procedures
for Biological Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Shane Gonen
6 Setting Up Parallel Illumination on the Talos Arctica for High-Resolution
Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Mark A. Herzik Jr
7 Multi-body Refinement of Cryo-EM Images in RELION. . . . . . . . . . . . . . . . . . . . 145
Takanori Nakane and Sjors H. W. Scheres
8 Evaluating Local and Directional Resolution
of Cryo-EM Density Maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Sriram Aiyer, Cheng Zhang, Philp R. Baldwin,
and Dmitry Lyumkis
9 Automated Modeling and Validation of Protein Complexes
in Cryo-EM Maps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Tristan Cragnolini, Aaron Sweeney, and Maya Topf
vii