توضیحاتی در مورد کتاب STRUCTURAL GENOMICS general applications.
نام کتاب : STRUCTURAL GENOMICS general applications.
ویرایش : 2
عنوان ترجمه شده به فارسی : کاربردهای کلی ژنومیک ساختاری
سری :
ناشر : SPRINGER-VERLAG NEW YORK
سال نشر : 2020
تعداد صفحات : 363
ISBN (شابک) : 9781071608920 , 1071608924
زبان کتاب : English
فرمت کتاب : pdf
حجم کتاب : 11 مگابایت
بعد از تکمیل فرایند پرداخت لینک دانلود کتاب ارائه خواهد شد. درصورت ثبت نام و ورود به حساب کاربری خود قادر خواهید بود لیست کتاب های خریداری شده را مشاهده فرمایید.
فهرست مطالب :
Preface
Contents
Contributors
Part I: Protein Production
Chapter 1: High-Throughput Protein Engineering by Massively Parallel Combinatorial Mutagenesis
1 Introduction
2 Materials
2.1 For Assembling a Barcoded Combinatorial Library by CombiSEAL
2.2 Sample Preparation for Barcode Sequencing
3 Methods
3.1 Creating Mutagenized Parts
3.1.1 Creating Mutagenized Parts Using a Library of Prebarcoded Storage Vectors
3.1.2 Creating Mutagenized Parts with Barcodes by PCR (an Alternative Strategy to Subheading 3.1.1 That Does Not Require the S...
3.2 Creating a Barcoded Combinatorial Protein Library
3.3 Amplification of the Plasmid Library
3.4 Expressing the Protein Library in Cell Culture for Screening
3.5 Preparing Samples for Barcode Sequencing
3.6 Barcode Sequencing Data Analysis
3.7 Epistasis Analysis
4 Notes
References
Chapter 2: Rational Design and Construction of Active-Site Labeled Enzymes
1 Introduction
2 Materials
2.1 Acquisition and visualization of the Enzyme´s Structural Information
2.2 Selection of Site for Fluorophore Attachment
2.3 Preparation of Cysteine Mutant
3 Methods
3.1 Protein Labeling with Fluoresecein-5-Maleimide
3.2 Validation of the Labeling Reaction
3.2.1 Qualitative Analysis by SDS-PAGE
3.2.2 Estimation of Degree of Labeling
4 Notes
References
Chapter 3: Screening and Production of Recombinant Human Proteins: Ligation-Independent Cloning
1 Introduction
2 Materials
2.1 PCR
2.2 Cloning
3 Methods
3.1 Construct Design
3.2 Primer and Plate Design
3.3 PCR
3.4 Vector Preparation
3.5 T4 DNA Polymerase Treatment
3.6 Annealing and Transformation
3.7 Colony PCR Screening
3.8 Preparation of Glycerol Stocks and 96-Well Miniprep
4 Notes
References
Chapter 4: Screening and Production of Recombinant Human Proteins: Protein Production in E. coli
1 Introduction
2 Materials
2.1 Transformation and Test Expression
2.2 Test Purification
2.3 Large-Scale Expression
2.4 Protein Extraction and Large-Scale Purification
3 Methods
3.1 Transformation into E. coli BL21(DE3)-R3-pRARE2
3.2 Test Expression
3.3 Test Purification
3.4 Large-Scale Expression
3.5 Protein Extraction
3.6 Large-Scale Protein Purification
3.7 Quality Assurance
4 Notes
References
Chapter 5: Screening and Production of Recombinant Human Proteins: Protein Production in Insect Cells
1 Introduction
2 Materials
2.1 Transposition and Bacmid Preparation
2.2 Transfection and Cell Growth
2.3 Virus Amplification and Test Expression
2.4 Test Purification
2.5 Large-Scale Expression
2.6 Protein Extraction and Large-Scale Purification
3 Methods
3.1 Transposition in E. coli DH10Bac or DH10EMBacY
3.2 Bacmid Production
3.3 Bacmid PCR Screen
3.4 Growth and Maintenance of Insect Cell Lines
3.5 Reviving Sf9 Cell Line from Frozen Stock
3.6 Suspension Culture of Sf9 Cells in Shake Flask
3.7 Cell Freezing
3.8 Decontamination and Cleaning of Shake Flasks
3.9 Transfection into Sf9 Cells
3.10 Virus Amplification and Test Expression
3.11 Test Purification
3.12 Virus Amplification
3.13 Large-Scale Expression
3.14 Protein Extraction
3.15 Large-Scale Protein Purification
3.16 Quality Assurance
4 Notes
References
Chapter 6: Expression Screening of Human Integral Membrane Proteins Using BacMam
1 Introduction
2 Materials
2.1 Transposition, Bacmid Preparation, and Transfection
2.2 Virus Amplification, Transduction, and Test Expression in Expi293F and HEK293S GnTI- Cells
2.3 Test Purification
2.4 Dionex Fluorescence Size Exclusion Chromatography (FSEC) Screening (Fig. 2)
3 Methods
3.1 Transposition, Bacmid Production, and Bacmid PCR Screen
3.2 Growth and Maintenance of Expi293F and HEK293S GnTI- Cell Lines
3.3 Reviving Expi293F and HEK293S GnTI- Cell Lines from Frozen Stocks
3.4 Suspension Culture of Expi293F and HEK293S GnTI- in Shake Flask
3.5 Cell Freezing
3.6 Decontamination and Cleaning of Shake Flasks
3.7 Transfection into Sf9 Cells
3.8 Test Expression (Transduction) in Expi293F or HEK293S GnTI- Cells
3.9 Test Purification
3.10 Dionex Fluorescence Size Exclusion Chromatography (FSEC) Screening
4 Notes
References
Chapter 7: High-Throughput Expression Screening in Mammalian Suspension Cells
1 Introduction
2 Materials
2.1 HEK293F Cell Maintenance
2.2 HEK293F Cell 24-Well Block Transfection
2.3 Expression Screening by Dot Blot
3 Methods
3.1 HEK293F Cell Maintenance
3.2 HEK293F Cell 24-Well Block Transfection
3.3 HEK293F Cell 96-well Block Transfection
3.4 Expression Screening by Dot Blot
4 Notes
References
Chapter 8: In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies
1 Introduction
2 Materials
2.1 E. coli Extracts Preparation (S30 Extract)
2.2 T7 RNA Polymerase Expression and Purification
2.3 Cell-Free Expression of Proteins
2.4 In Vitro Production of Perdeuterated protein in H2O
3 Methods
3.1 E. coli Extracts Preparation (S30 Extract)
3.2 T7 RNA polymerase Expression and Purification
3.3 Cell-Free Expression of Proteins
3.4 In Vitro Production of Perdeuterated Protein in H2O
3.4.1 Inhibition of Transaminases from S30 Extracts
3.4.2 In Vitro Synthesis of Perdeuterated Protein
3.5 Application to the Production of Large Perdeuterated Proteins for NMR Investigations
4 Notes
References
Chapter 9: Minimizing Heterogeneity of Protein Samples for Metal Transporter Proteins Using SAXS and Metal Radioisotopes
1 Introduction
2 Materials
3 Methods
3.1 Gel Filtration
3.2 SAXS
3.3 Radioactive Labeling of each Fraction
4 Notes
References
Part II: Structural Analyses and Data Management
Chapter 10: Hydrogen-Deuterium Exchange Mass Spectrometry for Probing Changes in Conformation and Dynamics of Proteins
1 Introduction
2 Materials
2.1 Centrifugal Ultrafiltration Devices for Buffer Exchange
2.2 HDX Buffer
2.3 Quenching Buffer
2.4 Proteases
2.5 LC Trap Column
2.6 LC Analytical Column
2.7 LC Solvents
2.8 Instrumentation
2.9 MS-Compatible, Nonionic Detergent
3 Methods
3.1 Sample Preparation
3.2 Hydrogen-Deuterium Exchange (HDX) and Quenching
3.3 Protease Digestion
3.3.1 Online Digestion
3.3.2 Offline Digestion
3.4 Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis
4 Notes
References
Chapter 11: BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy
1 Introduction
2 Materials
3 Methods
3.1 Sample Preparation
3.2 Wavelength Range, Baseline Subtraction, and Data Normalization
3.3 Single Spectrum Analysis
3.4 Fold Recognition
3.5 Multiple Spectra Analysis
3.6 Secondary Structure Composition from PDB Structures
3.7 Limitations of the BeStSel Method
4 Notes
References
Chapter 12: Navigating the Global Protein-Protein Interaction Landscape Using iRefWeb
1 Introduction
2 iRefIndex Consolidation
2.1 Summary of the iRefIndex Consolidation Procedure
2.2 iRefIndex Release V.16
3 Materials
4 Methods
4.1 Search for PPIs Involving Specific Proteins
4.2 Refine the Search Using Additional Proteins and Filters
4.3 Examine the Differences in Annotations from Different Source Databases
5 Notes
References
Chapter 13: State-of-the-Art Data Management: Improving the Reproducibility, Consistency, and Traceability of Structural Biolo...
1 Introduction
2 Data Model, Data Acquisition, and Validation
2.1 Data Model
2.2 Acquisition of Various Types of Laboratory Data
2.2.1 Reagents Module
Storage System
2.2.2 Protein Production Module
2.2.3 Crystallization Module
2.2.4 Structure Determination Component
2.2.5 Biochemical Experiments
Thermal Shift Assays
Isothermal Titration Calorimetry
Other Biochemical Assays
2.3 Data Validation
3 Technical Implementation
4 Data Analysis and Data Mining Tools
5 User Experience and PI Perspective
6 Enhancing Reproducibility and Efficiency of Experiments: Case Studies
7 Future Directions: Toward a Configurable LIMS Architecture
7.1 Data Model
7.2 System Architecture
7.3 Workflow Management
8 Conclusions
References
Part III: Modeling, Simulation, and Visualization
Chapter 14: Protein Structure Modeling with MODELLER
1 Introduction
2 Materials
3 Methods
3.1 Fold Assignment
3.2 Sequence-Structure Alignment
3.3 Model Building
3.4 Model Evaluation
3.5 Use of Multiple Templates
3.6 External Assessment
3.7 Structures of Complexes
4 Notes
References
Chapter 15: Parameterization of a Dioxygen Binding Metal Site Using the MCPB.py Program
1 Introduction
2 Materials
2.1 Preparation of the PDB File
2.2 Preparation of the mol2 Files
2.3 Create a MCPB.py Input File
3 Methods
3.1 Perform the First Step of the MCPB.py Protocol
3.2 Quantum Calculations by Gaussian16
3.3 Perform the Remaining Steps of the MCPB.py Protocol
3.4 Perform the LEaP Step and Check the Generated Files
3.5 Convert the AMBER Topology and Coordinate Files to the GROMACS Format
3.6 Perform MD simulations in GROMACS and Analyze the Results
4 Notes
References
Chapter 16: Parameterization of Large Ligands for Gromacs Molecular Dynamics Simulation with LigParGen
1 Introduction
2 Software
2.1 Web Server
2.2 Installed on Local Linux Computer
2.3 Commerical Software (Windows)
3 Protein Model
4 Ligand Model
4.1 Preparation of a Complete Geometric Model
4.2 Manual Rebuilding of Structural Model
5 Ligand Parameterization
5.1 Generating Parameters in Two Parts
5.2 Merging Geometry Parameters (.gro)
5.3 Combining Topology Parameters (.itp)
5.4 Rebuilding Coordinates (.pdb)
6 Running the MD Simulation (Using the Parameters)
6.1 Preparation
6.2 Energy Minimization (See Notes 10-12)
6.3 Equilibration, Production (See Notes 10-12)
7 Notes
References
Chapter 17: Simulation of Proteins Modified with a Fluorescent Label
1 Introduction
2 Software and Protein Structure
2.1 Software
2.2 Protein Structure
3 Fluorescent Probe Structure Building and Optimization
4 Parameterization of the Fluorescent Label Using ACPYPE
5 Incorporation of the Fluorophore Parameters into the CHARMM27 Force Field
5.1 Setting Up the Force Field
5.2 forcefield.doc
5.3 residuetypes.dat
5.4 aminoacids.rtp
5.5 aminoacids.hdb
5.6 ffbonded.itp
5.6.1 Bonds
5.6.2 Angles
5.6.3 Proper and Improper Dihedral Angles
6 Structure Construction of Protein-Fluorophore Complex
7 Molecular Dynamics Simulation of Fluorescent Probe-Labeled Protein
7.1 Generate Topology File
7.2 Topology
7.3 Define the Box
7.4 Add Water Molecules
7.5 Add Ions
7.6 Energy Minimization
7.7 Canonical Equilibration (NVT)
7.8 Gibbs Equilibration (NPT)
7.9 MD Simulation
8 Common Errors
8.1 Atom Name: O1, O2, OC1, OC2
8.2 Misidentification of Atom or Bond Types in ACPYPE
8.3 No Default U-B/Improper Dih. Types
8.4 The Sum of the Two Largest Charge Group Radii Is Larger than rlist
9 Notes
Appendix A: Proper and Improper Angle (See Figs. 1 and 2)
Appendix B: npt
Appendix C: topolpatch
topolpatch.pl
References
Chapter 18: Protocol for Simulations of PEGylated Proteins with Martini 3
1 Introduction
2 Martini Parameters for PEGylated Proteins
2.1 Software Requirements
2.2 Martini Parameters for Simple Proteins
2.2.1 Martinizing Lysozyme
2.2.2 Checking Protonation States
2.3 Martini Parameters for PEG
2.3.1 Homopolymer
2.3.2 End Group and Block-Copolymers
2.4 Linking PEG to the Protein
2.4.1 Designing Martini Parameters for the Linker
2.4.2 Combining Protein, Linker and PEG Parameters
2.5 Generation of Input Structures
3 Setting Up and Running a Simulation
4 Notes
References
Chapter 19: Molecular Data Visualization on Mobile Devices: A Quick Starter´s Guide
1 Introduction
2 Graphics Software
2.1 Hardware Used for Testing
2.2 Comparison of Graphics Software
3 Methods
3.1 Installation
3.2 Importing a PDB Entry
3.3 Viewing with Different Styles
3.3.1 Rainbow Coloring (Blue to Red) from N- to C-Termini
3.3.2 Transparent Items
3.3.3 Molecular Surface
3.4 Exporting an Image
4 Conclusion
5 Notes
Chapter 20: Molecular Data Visualization with Augmented Reality (AR) on Mobile Devices
1 Introduction
2 Hardware and Software Used for Testing
3 A Tutorial of Ollomol, a Standalone App
3.1 Ollomol Installation
3.2 Generating a Tracker Pattern for a PDB Entry
3.3 Viewing the Molecular Structure with AR in Ollomol
4 A Tutorial of WebAR-PDB, a Web-Server Application
4.1 Make the Cube Marker
4.2 PDB File Download
4.3 Viewing the Molecular Structure with AR
5 Discussion
6 Notes
References
Index