Contents
Chapter 1 Practical Approaches for In Situ X-ray Crystallography:
from High-throughput Screening to Serial Data Collection 1
Isabelle Martiel, Vincent Olieric, Martin Caffrey and
Meitian Wang
1.1 Introduction 1
1.1.1 What Exactly Is In Situ? 1
1.1.2 Goals of In Situ Experiments 3
1.1.3 Challenges of In Situ Methods 4
1.1.4 Enabling Technologies 5
1.2 In Situ Screening at the Synchrotron: Standard
SBS Plates 7
1.2.1 Development History 7
1.2.2 Plate Handling Hardware 9
1.2.3 Plate Optimization for In Situ 9
1.2.4 Automation and Pipeline Integration 10
1.3 Further Developments: Scale Reduction and
Microfluidics 11
1.3.1 Small Formats 11
1.3.2 Microfluidic Methods for In Situ 13
1.4 The Emergence of Serial In Situ Data Collection 15
1.4.1 Thin-film Sandwiches 15
1.4.2 Liquid Manipulation Methods 19
1.5 Conclusion and Outlook 20
Acknowledgements 20
References
Chapter 2 Delivery of GPCR Crystals for Serial Femtosecond
Crystallography 28
E. E. Abola, U. Weierstall, W. Liu and V. Cherezov
2.1 Introduction 28
2.2 Process Overview 30
2.3 Achieving Major Milestones 31
2.3.1 Large-scale Production of Stable Receptor
Constructs 31
2.3.2 Crystallization of Receptor Constructs 38
2.3.3 SFX Data Collection 39
2.4 Summary of Successful GPCR Structural Studies
at XFELs 42
Acknowledgements 46
References 46
Chapter 3 The Mesh&Collect Pipeline for the Collection of Multicrystal
Data Sets in Macromolecular Crystallography 54
Nicolas Foos, Gleb Bourenkov, Gordon Leonard, Igor
Melnikov, Christoph Mueller-Dieckmann, Max Nanao,
Alexander Popov, Gianluca Santoni and Ulrich Zander
3.1 Introduction 54
3.2 Dozor 59
3.3 Hierarchical Cluster Analysis (HCA) 59
3.4 Mesh&Collect in Practice 60
3.4.1 Solving the Crystal Structures of Membrane
Proteins with Very Small Crystals 61
3.4.2 Multi-crystal Data Collection for Ligand
Binding Studies 63
3.4.3 De novo Structure Solution using
Mesh&Collect 64
3.4.4 Mesh&Collect for the I-SAD/I-SIRAS
Solutions of the Crystal Structure of the KR2
Light-Driven Sodium Pump 68
3.4.5 Mesh&Collect at Room Temperature 69
3.5 The Pitfalls of HCA 69
3.6 ccCluster 75
3.7 Merging of Partial Data Sets Using Genetic
Algorithms 75
3.7.1 Grouping Partial Data Sets into
Chromosomes 77
3.7.2 Fitness Evaluation 77
……
8.5.3 Hit Finding 195
8.5.4 Bragg Diffraction Analysis: Indexing,
Merging, Post-refinement 197
8.5.5 Phasing and Model Refinement 202
8.5.6 De novo Phasing of SFX Data 203
8.5.7 SFX Data Volumes and Data Sharing 205
8.6 New Developments 206
8.6.1 Sparse Crystal Pattern Indexing 206
8.6.2 Nanocrystal Shape Transform Phasing 206
8.6.3 Continuous Diffuse Scattering 206
8.6.4 Single-layer 2D Crystals 207
8.6.5 Incoherent Diffractive Imaging 208
8.7 Conclusion 208
Acknowledgements 209
References 209
Subject Index 225
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