Structural Proteomics: High-Throughput Methods

✍ Scribed by Raymond J. Owens

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 350
• Series: Methods in Molecular Biology, 2305
• Edition: 3
• Category: Library

✦ Synopsis

This updated and expanded volume reflects the current state of the structural protein field with improved and refined protocols that have been applied to particularly challenging proteins. Beginning with a section on structural bioinformatics, the book continues with sections covering the challenge of producing high quality samples for structural studies, particularly mammalian membrane proteins and protein complexes, as well as protocols for structure determination, including the use of electrons in structural biology and more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Authoritative and up-to-date, Structural Proteomics: High-Throughput Methods, Third Edition will aid researchers in expanding our knowledge of this vital and expansive area of protein science.

Chapter 9 is available open access under a CC BY 4.0 license.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Structural Bioinformatics
Chapter 1: The Protein Data Bank Archive
1 Introduction
2 History of Macromolecular Structure Archiving
3 Role of the Worldwide Protein Data Bank in 3D Structure Data Archiving
4 Data Standards for 3D Biostructure Data Archiving
5 3D Biostructure Data Deposition, Biocuration, Validation, and Distribution
6 State of the Protein Data Bank Archive
7 Distributed Data Dissemination and Value-Added Annotations
8 Future of Structural Biology and the Role of the Worldwide PDB
References
Chapter 2: Computational Methods for the Elucidation of Protein Structure and Interactions
1 Introduction
2 A Brief Summary of Protein Classification and Data Repositories
3 Types of Structure Prediction; Comparative Versus Ab Initio Modeling
4 Stages in Template-based Modeling (TBM)
4.1 Sequence Alignment and Template Identification
4.2 Loop Identification and Side-Chain Packing
4.3 QA and Ranking Models
4.4 Refinement
4.5 What if your Model Is Not a Good One?
4.6 Disorder and Secondary Structure Prediction
4.7 Distant Homology Searches, Fold Recognition, and Threading Programs
4.8 Ab Initio or (Template) Free Modeling Methods
5 Comprehensive or Integrated Structure Prediction Webservers
5.1 IntFOLD
5.2 I-TASSER
5.3 Phyre2
5.4 Robetta
5.5 Swiss-Model
6 CASP and CAMEO
7 Protein-Protein Interactions (PPI) and Quaternary Structure Prediction
7.1 Docking Programs
7.2 The Evolution of Docking Methods
8 Notes
References
Chapter 3: Methods for Molecular Modelling of Protein Complexes
1 Introduction
2 Modelling Protein-Small Molecule Complexes
2.1 Selecting the Small Molecule Library
2.2 Predicting Small Molecule Binding Pockets on the Target Protein
2.3 Docking Small Molecules on a Target Protein: Sampling the Ligand Conformation and Scoring
2.4 Shortlisting the Compounds
3 Modelling Protein-Peptide Complexes
3.1 Predicting Binding Sites for Peptide Ligands
3.2 Modelling Protein-Peptide Complexes
3.2.1 Predicting the Sequence of the Peptides and the Structure of the Protein-Peptide Complex
3.2.2 Docking Peptides onto Target Proteins
Template-Based Docking of Protein-Peptide Complexes
Local Docking of Protein-Peptide Complexes
Blind Docking of Protein-Peptide Complexes
3.3 Assessing Predicted Models with Various Scoring Schemes
4 Modelling Protein-Protein Complexes
4.1 Template-Based Prediction of Structure of a Protein-Protein Complex Given Structures of the Target Proteins
4.2 Template-Based Prediction of Structure of a Protein Complex When the Structures of the Constituent Target Proteins Are Not...
4.3 Protein-Protein Docking
4.3.1 Restraint-Based Local Docking of Protein-Protein Complexes
4.3.2 Blind Docking of Protein-Protein Complexes
4.4 Evaluating Protein-Protein Complexes
5 Modelling Protein-Nucleic Acid Complexes
5.1 Template-Based Modelling of Protein-Nucleic Acid Complexes
5.1.1 Homology Modelling of Protein-Nucleic Acid Complexes
5.1.2 Fragment-Based Modelling of Protein-Nucleic Acid Complexes
5.2 Docking of Protein-Nucleic Acid Complexes
5.2.1 Knowledge-Based Docking of Protein-Nucleic Acid Complexes
5.2.2 Blind Docking of Protein-Nucleic Acid Complexes
6 Modelling Macromolecular Assemblies Containing Various Biomolecules
6.1 Data Collection
6.2 Data Representation and Model Evaluation
6.3 Sampling and Optimization
6.4 Ensemble Analysis
7 Notes
References
Part II: Protein Production
Chapter 4: High-Level Production of Recombinant Eukaryotic Proteins from Mammalian Cells Using Lentivirus
1 Introduction
1.1 Lentiviral Transduction of HEK293 Cells
1.2 Brief Overview of the Procedure
1.3 Biological Safety
2 Materials
2.1 Mammalian Cell Lines and Bacterial Cloning Strains
2.2 Lentiviral Plasmids
2.3 Tissue Culture Reagents
2.4 Equipment
3 Methods
3.1 Safety Guidelines
3.2 General Cell Culture
3.3 Plasmid Preparation
3.4 Lentivirus Production and Transduction of Expression Cells
3.4.1 Production of Lentiviral Particles
3.4.2 Transduction of Expression Cells
3.5 Determination of Functional Lentiviral Titer by Flow Cytometry
3.6 Determination of Functional Lentiviral Titer by Endpoint Dilution and Fluorescence Microscopy
3.7 Protein Expression in Adherent Polyclonal Stable Cell Lines
3.8 Protein Expression in Suspension-adapted Polyclonal Stable Cell Lines
4 Notes
References
Chapter 5: Transient Transfection and Expression of Eukaryotic Membrane Proteins in Expi293F Cells and Their Screening on a Sm...
1 Introduction
2 Materials
2.1 Sub-cloning Genes of Interest in pOPIN Vectors Using In-Fusion Cloning Technique
2.2 Preparation of Transfection-Grade Plasmids
2.3 Transient Transfection and Expression in Expi293F Cells: General Procedure
2.4 Screening of Expression Parameters on a Small Scale: 1 mL/12-Well Plate Expression
2.5 Up-Scaled Expression
2.6 Visualization of Expression by Fluorescence Microscopy
2.7 Analysis of Expression with Tali Imaging System
2.8 Analysis of Expression Using In-Gel GFP Fluorescence of Cell Probes
2.9 Membranes Preparation
2.10 Screening of Detergents for Protein Extraction from Membranes on a Small Scale
2.11 Purification of His-Tagged Targets in Different Detergents on a Small Scale
2.12 Purification of Strep-Tagged Targets in Different Detergents on a Small Scale
2.13 Quality Control of His- and Strep-Tag-Purified Samples: FSEC
2.14 Purification from Small-Scale Expression Tests (3 mL/6-Well Plate)
2.15 Large-Scale Affinity Purification
3 Methods
3.1 Sub-cloning Genes of Interest in pOPIN Vectors Using In-Fusion Cloning Technique
3.2 Preparation of Transfection-Grade Plasmids
3.3 Transient Transfection and Expression in Expi293F Cells: General Procedure
3.4 Screening of Expression Parameters on a Small Scale: 1 mL/12-Well Plate Expression
3.5 Up-Scaled Expression
3.6 Visualization of Expression by Fluorescence Microscopy
3.7 Analysis of Expression with Tali Imaging System
3.8 Analysis of Expression Using In-Gel GFP Fluorescence of Cell Probes
3.9 Membranes Preparation
3.10 Screening of Detergents for Protein Extraction from Membranes on a Small Scale
3.11 Purification of His-Tagged Targets from Membranes in Different Detergents on a Small Scale
3.12 Purification of Strep-Tagged Targets from Membranes in Different Detergents on a Small Scale
3.13 Quality Control of His- and Strep-Tag Purified Samples: FSEC
3.14 Purification from Small-Scale Expression Tests (3 mL/6-Well Plate)
3.15 Large-Scale Affinity Purification
4 Notes
References
Chapter 6: Reproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells
1 Introduction
2 Materials
2.1 Expression Vectors and Cell Lines
2.2 Cell Culture
2.3 Transfection Reagents and Additional Chemicals for Protein Production
3 Methods
3.1 Construction of Expression Vectors
3.2 Transient Expression in Hi5 Cells
3.3 Production of the SARS-CoV-2 S1-Opt-delFurin-hFc
3.3.1 Expression and Purification of S1-Opt-delFurin-hFc
3.3.2 SEC-MALS Analysis of the Glycosylated S1-Opt-delFurin-hFc Protein
3.3.3 Enzyme-Linked Immuno Sorbent Assay (ELISA)
3.4 Analysis of Glycosylation
4 Notes
References
Chapter 7: SynBac: Enhanced Baculovirus Genomes by Iterative Recombineering
1 Introduction
2 Materials
2.1 In Silico Design
2.2 Preparation of DNA Fragment of Choice
2.3 Bacterial Cells Containing BAC and Plasmid pRed/ET
2.4 First Recombination Reaction
2.5 Second Recombination Reaction
2.6 Removal of Selection Marker Res2
3 Methods
3.1 In Silico Design
3.2 Preparation of the DNA Fragment of Choice
3.3 Bacterial Cells Containing BAC and Plasmid pRed/ET
3.4 First Red/ET Recombination
3.5 Second Recombination Reaction
3.6 Removal of Selection Marker Res2
4 Notes
References
Chapter 8: Gene Tagging with the CRISPR-Cas9 System to Facilitate Macromolecular Complex Purification
1 Introduction
2 Materials
2.1 Oligonucleotides and Plasmids
2.2 Cell Culture
2.3 Nucleofection AMAXA
2.4 Nano-Glo HiBiT Detection System
2.5 Ouabain Selection, Pool and Clone Analysis
2.6 Biochemical Characterization and Purification
3 Methods
3.1 Design of sgRNAs for Targeting the Gene of Interest and Cloning into Guide RNA Expression Plasmid
3.2 Design of the Single-Stranded Oligonucleotide (ssODN) Donor
3.3 Optimal sgRNA Selection with the Nano-Glo HiBiT Lytic Detection System
3.4 Transfection of Gene Editing Reagents into K562 Cells and Ouabain Selection
3.5 Isolation of Gene-Edited Cells by Single-Cell Cloning
3.6 Validation of Gene Tagging by Western Blot
3.6.1 Preparation of Cell Pellets
3.6.2 Analysis of Tagged Proteins in the Whole Cell Extract
3.6.3 Analysis of the Target Complex after Immunopurification
3.7 Suspension Cultures and Purification
3.7.1 Large-Scale Suspension Cultures
3.7.2 Immunopurification
4 Notes
References
Chapter 9: Synthesis of Fluorescently Labeled Antibodies Using Non-Canonical Amino Acids in Eukaryotic Cell-Free Systems
1 Introduction
2 Materials
2.1 CFPS Using CHO Lysate
2.2 Preparation of Orthogonal Synthetase
2.3 Preparation of Orthogonal tRNA
2.3.1 PCR Amplification of the tRNA Gene
2.3.2 Transcription, Isolation, and Folding of tRNA
2.4 Site-Specific Incorporation of Non-canonical Amino Acids
2.5 Qualitative Protein Analysis
3 Methods
3.1 Cell-free Synthesis and Fluorescence Labeling of IgG Using Pre-Charged tRNA
3.1.1 Batch-Based CFPS
3.2 Fractionation of Translation Mixtures
3.3 Cell-free Synthesis and Fluorescence Labeling of IgG Using Orthogonal System
3.3.1 Preparation of Orthogonal Synthetase
3.3.2 Preparation of Orthogonal tRNA
3.3.3 Cell-free Synthesis of IgG and Site-Specific Incorporation of Non-canonical Amino Acids (Ncaa)
3.3.4 Labeling of IgG with Fluorescent Dye (Staudinger Ligation)
3.4 Qualitative Protein Analysis
3.4.1 SDS-PAGE and Autoradiography
3.4.2 In-Gel Fluorescence Analysis and Autoradiography
4 Notes
References
Part III: Structure Determination
Chapter 10: Solid-State NMR Spectroscopy for Studying Microtubules and Microtubule-Associated Proteins
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Preparation of Isotope-Labeled MTs
2.3 Purification of Isotope-Labeled CKK Domain from Bacteria
3 Methods
3.1 Suspension Cell Culture for Isotope Labeling
3.2 Sample Preparation of Isotope-Labeled MTs (See also ref)
3.3 Purification of Isotope-Labeled CKK from Bacteria
3.4 ssNMR and DNP Experiments on MTs and MT-MAP Complexes
4 Notes
References
Chapter 11: Dynamic Structural Biology Experiments at XFEL or Synchrotron Sources
1 Introduction
2 Sample Preparation (See Note 1)
2.1 Free Interface Diffusion (FID)
2.2 Batch Method
3 Sample Delivery and Data Collection
3.1 Photosensitive Systems
3.1.1 Photosystem II as an Example System for Time-Resolved SFX
3.1.2 Caged Protein
3.2 Enzyme Catalysis
3.2.1 Mix-Inject Sample delivery for Time-Resolved SFX
3.3 ``Multimessenger techniques´´ Help Reduce Ambiguity in Time-Resolved SFX Experiments
4 Notes
References
Chapter 12: From Tube to Structure: SPA Cryo-EM Workflow Using Apoferritin as an Example
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Software
3 Methods
3.1 Preparation of Sample Grids
3.1.1 Glow Discharge
3.1.2 Vitrification
3.1.3 Prepare the Cryogen Cup
3.1.4 Plunge Grids
3.1.5 Clipping
3.1.6 Loading Cassette
3.2 Microscopy
3.2.1 Microscope Alignment and Performance Check (Also See Videos on EM-learning.com)
3.2.2 Grid Screening and Setup of Automated SPA Data collection Using EPU
3.2.3 Data Processing
Motion Correction
CTF Estimation
Live Processing
Particle Picking
2D Classification
Initial Model Generation
3D Classification
3D Refinement
Post-Processing and Other Corrections
4 Notes
References
Chapter 13: Image Processing in Cryo-Electron Microscopy of Single Particles: The Power of Combining Methods
1 Introduction
2 Methods
2.1 From Frames to Valid Micrographs
2.2 Finding Particles in Micrographs
2.3 2D Classification
2.4 Constructing an Initial Map
2.5 Achieving an Homogeneous Population
2.6 Refining an Homogeneous Population
2.7 Final 3D Reconstruction with Consensus Geometry
2.8 Post-Processing
2.9 Validation
2.10 Interpretation and Model Building
3 Conclusions
References
Chapter 14: Setup and Troubleshooting of Volta Phase Plate Cryo-EM Data Collection
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 15: Cryo-Focused Ion Beam Lamella Preparation Protocol for in Situ Structural Biology
1 Introduction
2 Materials
2.1 Preparation and Maintaining of the Mammalian Adherent Cell Culture
2.2 Preparation and Maintaining of Saccharomyces cerevisiae Cell Culture
2.3 Protein Crystallization
2.4 Preparation and Vitrification of TEM Grids
2.5 Mounting TEM Grids into the AutoGrid
3 Methods
3.1 Preparation and Maintaining of the Mammalian Adherent Cell Culture
3.2 Preparation of TEM Grids with Mammalian Adherent Cells
3.3 Vitrification of the Mammalian Adherent Cells
3.4 Cultivation of Saccharomyces cerevisiae Suspension Cell Culture
3.5 Vitrification of Saccharomyces cerevisiae Cells on EM Grids
3.6 Preparation of Protein Crystal Samples for Cryo-FIBM Workflow
3.7 Vitrification of Proteinase K Crystals on EM Grids
3.8 Mounting TEM Grids into the AutoGrid
3.9 Sample Manipulation in FIB-SEM Microscope
3.10 SEM Imaging of the Specimen and Grid Quality Control before FIB Milling
3.11 Preparation of Cellular or Protein Crystal Lamella
4 Notes
References
Chapter 16: Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED
1 Introduction
2 Materials
2.1 Preparing Crystals for MicroED
2.2 Identification of Microcrystals
2.3 Grid Preparation
2.4 Data Collection and Processing
3 Methods
3.1 Preparing Crystals for MicroED
3.1.1 Fragment Crystals by Pipetting
3.1.2 Fragment Crystals by Sonication
3.1.3 Fragment Crystals by Vortexing
3.1.4 FIB Milling
3.2 Identification of Microcrystals
3.2.1 Identification of Microcrystals Using Negative Stain EM
3.3 Grid Preparation for MicroED
3.3.1 Glow Discharge
3.3.2 Automated Vitrification
3.3.3 Manual Blotting and Freezing
3.4 Setting up the Microscope (ThermoFisher Cryo-TEMs Such as Glacios, Talos Arctica, and Titan Krios)
3.4.1 Calibrating the Electron Dose of the Microscope
3.4.2 Calibrating Detector Distance
3.4.3 Setting up the Microscope for Low-Dose Electron Diffraction (Talos Arctica)
3.5 Grid Screening
3.5.1 Atlas
3.5.2 Grid Screen in Search Mode
3.6 Data Collection
3.7 Data Processing
3.7.1 Indexing and Integration with iMosflm
3.7.2 Indexing and Integration with XDS
3.7.3 Merging and Phasing Preparation
3.7.4 Merging iMosflm Processed Data with AIMLESS
3.7.5 Merging XDS Processed Data with XSCALE
3.7.6 Molecular Replacement Using MOLREP
3.7.7 Molecular Replacement Using Phaser
3.7.8 Refinement Using REFMAC
3.7.9 Refinement with phenix.refine
4 Notes
References
Index