Advanced Technologies for Protein Complex Production and Characterization: Volume II (Advances in Experimental Medicine and Biology, 1453)

Preface
	References
Contents
1: Immunoprecipitation Methods to Isolate Messenger Ribonucleoprotein Complexes (mRNP)
	1.1 Introduction
	1.2 Endogenous mRNP Purification Using RNA Immunoprecipitation Methods
		1.2.1 Native Immunopurification
			1.2.1.1 Key Requirements for RIP
			1.2.1.2 mRNA Identification and Validation of the RIP Experiment
		1.2.2 Epitope Tagging and RIP
		1.2.3 Formaldehyde Cross-linking and RIP
		1.2.4 UV Cross-linking and RIP
	1.3 Cell-Free Extract mRNP Immunoprecipitation Methods
		1.3.1 GST Pull-Down Experiments Using Total RNA from Cell Extracts
		1.3.2 Crosslinking of Total Cell Extracts with In Vitro Transcribed 4-thioU or 6-thioG Labelled mRNAs
	1.4 Direct mRNA IP: Anti-m6A, m5C and Anti-TMG IP
		1.4.1 m6A Me and m5C RIP
		1.4.2 Trimethylguanosine-Capped mRNA Immunoprecipitation (TMG-IP)
	1.5 Conclusion
	References
2: Purification of In Vivo or In Vitro-Assembled RNA-Protein Complexes by RNA Centric Methods
	2.1 Introduction: Issues and Challenges of RNP Purifications
	2.2 First Affinity Purification Methods
	2.3 Affinity Purification Based on Artificially Selected or Natural RNA Motifs
		2.3.1 Antibiotic-Binding RNA Aptamers
		2.3.2 Streptavidin and Sephadex Aptamers
		2.3.3 Affinity Purification with RNA Tags Derived from Natural Sequences
	2.4 RNP Capture Based on Antisense Biotinylated Oligonucleotides
	2.5 RNP Purification Based on Ligation of a DNA-Oligonucleotide Tag
		2.5.1 Tagging RNA by Splint-Dependent Ligation with T4 DNA Ligase
		2.5.2 Direct Ligation of 5′-Adenylated Deoxyoligonucleotides Tag by T4 RNA Ligase 2
	2.6 Outlook
	References
3: Peptide-Based Mass Spectrometry for the Investigation of Protein Complexes
	3.1 Introduction
	3.2 Sample Preparation
		3.2.1 Sample Fractionation
		3.2.2 Reduction and Alkylation of Cysteine
		3.2.3 Protein Digestion
		3.2.4 Multiple Enzymatic Digestion
		3.2.5 Chemical Hydrolysis
	3.3 Sample Analysis
		3.3.1 LC-MS/MS
		3.3.2 MALDI-TOF-MS
		3.3.3 Mass Spectrometers and Data Acquisition
		3.3.4 Peptide Ionization
		3.3.5 Peptide Fragmentation
	3.4 Data Analysis
	References
4: Probing Protein Complexes Composition, Stoichiometry, and Interactions by Peptide-Based Mass Spectrometry
	4.1 Introduction
	4.2 Subunit Identification and Characterization
	4.3 Quantitative Composition: Stoichiometry and Number of Subunits
	4.4 Structural Investigation of Protein Complexes
		4.4.1 Cross-Linking and Mass Spectrometry
		4.4.2 Hydrogen Deuterium Exchange (HDX)
	References
5: Discovery and Characterization of Linear Motif Mediated Protein-Protein Complexes
	5.1 Introduction
	5.2 Experimental PPI Techniques for Linear Binding Motif Discovery
	5.3 Prediction of Linear Motifs In Silico
	5.4 Capture and Validation of Protein-Linear Motif Based Interactions
	5.5 Linear Motif Mediated PPI Dynamics
	5.6 Conclusion
	References
6: Protein-Protein Binding Kinetics by Biolayer Interferometry
	6.1 Biolayer Interferometry
		6.1.1 Binding Experiments
		6.1.2 Quantitation Experiments
		6.1.3 BLI in Comparison with Other Techniques
	6.2 BLI Assay Development
		6.2.1 Prepare Buffers, Regeneration Solution, Ligand, and Analyte Samples
		6.2.2 Hydrate the Biosensor in Assay Buffer to Minimize Nonspecific Signal
		6.2.3 Maintain a Stable Assay Temperature
		6.2.4 Immobilize the Ligand onto the Biosensor Surface
		6.2.5 Measure Association and Dissociation Kinetics
		6.2.6 Regenerate Sensor Tip Surface (Optional)
		6.2.7 Data Processing, Analysis, and Fitting
	6.3 Examples of Protein-Protein Interactions Studied by BLI
	6.4 Concluding remarks
	References
7: Studying Macromolecular Interactions of Cellular Machines by the Combined Use of Analytical Ultracentrifugation, Light Scat...
	7.1 Introduction
	7.2 Analytical Ultracentrifugation (AUC)
		7.2.1 Instrumentation and General Experimental Considerations
		7.2.2 Sedimentation Velocity (SV)
		7.2.3 Sedimentation Equilibrium (SE)
	7.3 Light Scattering (LS) Techniques
		7.3.1 Dynamic Light Scattering (DLS)
		7.3.2 Multi-angle Light Scattering (MALS)
			7.3.2.1 Size Exclusion Chromatography Coupled to Multi-angle Light Scattering (SEC-MALS)
			7.3.2.2 Composition Gradient Multi-angle Light Scattering (CG-MALS)
	7.4 Fluorescence Spectroscopy Approaches
	7.5 Global Application of these Techniques to the Study of Complex Formation
		7.5.1 Quantitative Analysis of the Self-Association and Activation of the Bacterial Chaperone ClpB
		7.5.2 Untangling the Central Bacterial Division Protein FtsZ Polymers
		7.5.3 Unravelling the Interactions of the Bacterial Division Protein FtsZ with the Membrane Anchor ZipA Solubilized in Nanodis...
		7.5.4 Protein-DNA Complexes: The Interaction Between the Repressor Protein Reg576 and Its DNA Operator
	References
8: The Complementarity of Nuclear Magnetic Resonance and Native Mass Spectrometry in Probing Protein-Protein Interactions
	8.1 Introduction
	8.2 Native MS Applications in Structural Biology
	8.3 Advantages and Limitations of Native MS
	8.4 NMR Applications in Determining Molecular Interactions
	8.5 Advantages and Limitations of NMR in Determining Interactions
	8.6 The Successful Combination of the Two Techniques
	8.7 Concluding Remarks and Outlook
	References
9: X-Ray Crystallography for Macromolecular Complexes
	9.1 Introduction
	9.2 Producing Multisubunit Complexes Discovery
	9.3 Crystallization and Crystal Handling
	9.4 X-Ray Diffraction Experiment and Data Processing
		9.4.1 Data Collection Strategies
		9.4.2 Multi-crystal and Microcrystal Mounts
		9.4.3 Multi-crystal Approaches and Serial X-Ray Crystallography
		9.4.4 X-Ray Free Electron Laser (XFEL)
	9.5 Phasing and Structure Determination
	9.6 Macromolecular Refinement
	9.7 Model Building
	9.8 Structure Analysis and Interpretation
	9.9 Power of Two: Combining XRD with SAXS and Cryo-EM
	9.10 Conclusions
	References
10: Characterization of Biological Samples Using Ultra-Short and Ultra-Bright XFEL Pulses
	10.1 Introduction
	10.2 Properties and Benefits of XFEL Sources
		10.2.1 Time Resolution
		10.2.2 Signal From Smaller Crystals
		10.2.3 Possibility to Follow Irreversible Time-Resolved Processes
	10.3 Effects of XFEL Radiation on Biological Samples
	10.4 Use of XFELs for Life Science Research
		10.4.1 Current State of the Art and ``Standard´´ Experimental Options
		10.4.2 Crystallography
		10.4.3 Single Particle (Coherent Diffraction) Imaging
		10.4.4 Scattering Techniques
		10.4.5 Complementarity with Synchrotron Sources
	10.5 How to Prepare an XFEL Experiment
	10.6 XFEL Experiment Simulation
	10.7 Sample Characteristics
		10.7.1 Crystal Size and Estimation of Amounts Needed
		10.7.2 Single Particle Sizes (Easy to Challenging) and Estimation of Amount Needed
		10.7.3 On-Site Sample Preparation and Biophysical Characterization
		10.7.4 Choice of Sample Injection Options
		10.7.5 Liquid Jets
		10.7.6 Mixing Jets
		10.7.7 Aerosol
		10.7.8 Droplet Injection
		10.7.9 Fixed Targets
	10.8 Serial Femtosecond Crystallography (SFX)
		10.8.1 Comparison with Single Crystal Crystallography
		10.8.2 Calibration of Raw Data
		10.8.3 Hit Finding - Identification of Frames with Usable Diffraction Data
		10.8.4 Indexing
		10.8.5 Merging
		10.8.6 Overview of Analysis Procedures
	10.9 Single Particle Imaging (SPI)
		10.9.1 Comparison with SFX
		10.9.2 Overview of Analysis Procedures
		10.9.3 Calibration of Raw Data
		10.9.4 Hit Finding - Identification of Single Particles Only
		10.9.5 Orientation Determination
		10.9.6 Phasing
	10.10 Notes on the Need for and Use of Automation
	References
11: Small-Angle X-Ray Scattering for Macromolecular Complexes
	11.1 Introduction
	11.2 Dilution Series SAXS
	11.3 Inline Purification SAXS - Fighting Polydispersity
	11.4 Buffer Considerations
	11.5 Analysis of Macromolecular Complexes
	11.6 Presenting ``Complex´´ Data for Publication
	11.7 Case Study
	11.8 Conclusions
	References
12: Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines
	12.1 Introduction
	12.2 Approaches for High-Quality Sample Preparation
		12.2.1 Selecting the Optimal Expression System
		12.2.2 Sample Quality Control and Buffer Optimization
		12.2.3 Improving Size and Stability: Nanobodies and Fusion Proteins
		12.2.4 Improving Stability: Polyproteins and Crosslinking
		12.2.5 Stabilizing Membrane Proteins
			12.2.5.1 New Amphiphilic Detergents
			12.2.5.2 Amphipathic Polymers: A Substitute for Detergent
			12.2.5.3 Nanodiscs Mimic the Lipid Bilayer
			12.2.5.4 Saposin-Based Nanoparticles and Styrene-Maleic Acid (SMA) Copolymers
	12.3 Cryo-grid Preparation and Ice Thickness
		12.3.1 New Grid Materials to Reduce Particle Movement
		12.3.2 Cryo-grid Preparation: From Pipetting-Blotting-Plunging Devices to New Automated Cryo-Grid Preparation Procedures
	12.4 Imaging In-Focus: The Volta Phase Plate
	12.5 Challenges and Outlook
	References
13: Characterization of Complexes and Supramolecular Structures by Electron Microscopy
	13.1 Introduction
	13.2 Recent Technical Advances
	13.3 Analysis of Macromolecular Complexes
		13.3.1 Purification Requirements
		13.3.2 Structure Determination: From Molecular Up to Atomic Resolution
		13.3.3 Localization of Ligands, Substrates and Small Molecules
		13.3.4 Study of Related Conformations and Snapshots of Dynamic Processes
	13.4 In Situ Structural Determination by Electron Tomography
		13.4.1 Sample Preparation for Cryo-Electron Tomography
		13.4.2 Improvement of Tomogram Interpretation and Resolution
	13.5 Conclusions
	References
Index