Cytoskeleton methods and protocols

Preface
Contents
Contributors
Part I: Imaging the Cytoskeleton
	Chapter 1: Super-Resolution Imaging of the Actin Cytoskeleton in Living Cells Using TIRF-SIM
		1 Introduction
			1.1 Super-Resolution Imaging of the Actin Cytoskeleton
			1.2 The Actin Cytoskeleton and Glucose-Stimulated Insulin Secretion (GSIS) in Pancreatic Beta Cells
			1.3 TIRF-SIM Microscopy
			1.4 Confocal Laser Scanning Microscopy with Airyscan 2 Technology
			1.5 Image Processing, Analysis, and Workflow
			1.6 The Future of Super-Resolution Microscopy
		2 Materials
			2.1 Cell Culture
			2.2 Transient Cell Transfection
			2.3 TIRF-SIM Live Cell Imaging
			2.4 CLSM (Zeiss LSM 980 with Airyscan 2) Live Cell Imaging
			2.5 Image Processing and Analysis
		3 Methods
			3.1 Cell Culture
			3.2 Transfection of INS-1 (832/13) Cells with Actin Expression Vector
			3.3 Super-Resolution Live Cell Imaging
				3.3.1 TIRF-SIM Microscopy
				3.3.2 Enhanced Resolution Confocal Laser Scanning Microscopy (CLSM)
				3.3.3 Image Processing and Analysis
					Automated Tracking of Motile Microvilli Using TrackMate
					Curvature Analysis of Cortical Actin Bundles Using B-Splines
					Quantification of Actin Filament Network Density
		4 Notes
		References
	Chapter 2: Imaging Cytoskeleton Components by Electron Microscopy
		1 Introduction
		2 Materials
			2.1 Cell Culture and Extraction
			2.2 Fixation
			2.3 Dehydration and Critical Point Drying (CPD)
			2.4 Platinum and Carbon Coating
			2.5 Preparation of Replicas
			2.6 Immunogold PREM
			2.7 Correlative PREM
			2.8 Actin Depletion
		3 Methods
			3.1 Cell Culture and Extraction
			3.2 Fixation
			3.3 Dehydration and  CPD
			3.4 Platinum and Carbon Coating
			3.5 Preparation of Replicas
			3.6 Immunogold PREM
			3.7 Correlative Light Microscopy and PREM (CLEM)
			3.8 Actin Depletion
		4 Notes
		References
	Chapter 3: Imaging the Actin Cytoskeleton in Live Budding Yeast Cells
		1 Introduction
			1.1 Choosing a Fluorescent Protein for Detection of Cellular Structures
			1.2 Tagging Endogenous Proteins
			1.3 Visualization of Actin Dynamics with Actin Bundling Protein Abp140p
			1.4 Visualization of Actin Dynamics with Actin-Binding Peptide Lifeact
		2 Materials
			2.1 Yeast Growth Media
			2.2 PCR Amplification
			2.3 Yeast Transformation and Marker Excision
			2.4 Wide-Field Imaging of Live Yeast
		3 Methods
			3.1 Modification of Yeast Genes at Their Chromosomal  Loci
				3.1.1 Primer Design
				3.1.2 Amplification of Insertion Cassette from Tagging Vector
			3.2 Lithium Acetate Transformation of Yeast
			3.3 Marker Excision by Cre Recombinase
			3.4 Validating and Characterizing Fluorescent Protein-Tagged Cytoskeletal Proteins
				3.4.1 PCR Screening and Sequencing
				3.4.2 Verification of Protein Expression and Function
			3.5 Wide-Field Imaging of Living Cells
			3.6 Multicolor Imaging
			3.7 Preparing Cells for General Short-Term Imaging
			3.8 Preparing Cells for Short Time-Lapse Imaging of Actin Cable Dynamics
			3.9 Optimizing Imaging Conditions and Preventing Toxicity
				3.9.1 Checking for Photodamage
				3.9.2 Reducing Phototoxicity and Photobleaching
				3.9.3 Increasing Signal-to-Noise Ratio
			3.10 Special Considerations for Quantitative Analysis
			3.11 Deconvolution of Wide-Field Fluorescence Images
				3.11.1 Volocity Deconvolution
				3.11.2 Fiji/ImageJ Deconvolution
			3.12 Quantification of Fluorescent Signals
				3.12.1 Using Volocity to Measure Integrated Intensity
				3.12.2 Using Volocity to Measure Mean Intensity
				3.12.3 Using Fiji/ImageJ to Measure Mean or Integrated Intensity
			3.13 Analysis of Actin Dynamics: Measuring Retrograde Actin Cable Flow Velocity
		4 Notes
		References
	Chapter 4: Imaging the Actin Cytoskeleton in Fixed Budding Yeast Cells
		1 Introduction
		2 Materials
			2.1 Yeast Growth Media
			2.2 Fixation and Staining of Yeast Cells
			2.3 Antibodies
			2.4 Equipment for Wide-Field Microscopy
		3 Methods
			3.1 Fixation of Yeast Cells
			3.2 Immunofluorescence
				3.2.1 Pretreatment of Antibodies with Yeast Cell Walls
				3.2.2 Preparation of a Staining Chamber
				3.2.3 Removal of the Cell Wall with Zymolyase
				3.2.4 Attachment of Spheroplasts to Coverslip with Polylysine
				3.2.5 Indirect Immunofluorescence Staining of Attached Spheroplasts
			3.3 Visualization of the Actin Cytoskeleton in Fixed Cells with Fluorescent Phalloidin
			3.4 Preparation for Super-Resolution Structured Illumination Microscopy (SR-SIM)
			3.5 Imaging Fixed Budding Yeast Cells: Wide-Field Microscopy and SR-SIM
				3.5.1 Visualization of Fixed Cells by Wide-Field Imaging and Deconvolution
					Deconvolution Using Volocity
					Deconvolution Using Fiji/Image  J
				3.5.2 Visualization of Fixed Cells by Super-Resolution Structured Illumination Microscopy (SR-SIM)
					Sample Preparation and Imaging
					Reconstruction
			3.6 Quantification of Fluorescent Signals
				3.6.1 Using Volocity to Measure Integrated Intensity
				3.6.2 Using Volocity to Measure Mean Intensity
				3.6.3 Using Fiji/ImageJ to Measure Mean or Integrated Intensity
				3.6.4 Quantitative Analysis of Actin Cable Number, Thickness, and Intensity Using Line Profile Measurements
					Creating Line Profiles
					Measurements of Cable Number, Thickness, and Intensity
		4 Notes
		References
	Chapter 5: Imaging of Actin Cytoskeletal Integrity During Aging in C. elegans
		1 Introduction
			1.1 Imaging of the Actin Cytoskeleton in C. elegans
			1.2 Making of C. elegans Transgenic Lines
			1.3 Form and Function of Actin During Aging in Live C. elegans
			1.4 Physiological Assays to Measure Organismal Health
		2 Materials
			2.1 C. elegans Growth Media
			2.2 MosSCI Construct Synthesis
			2.3 C. elegans Microinjections
			2.4 C. elegans Synchronization
			2.5 Live-Cell Imaging of C. elegans
		3 Methods
			3.1 Growth and Maintenance of C. elegans
			3.2 Making C. elegans MosSCI Strains
			3.3 MosSCI Construct Design and Synthesis
			3.4 C. elegans Microinjections and MosSCI Selection
				3.4.1 Preparation of Injection  Pads
				3.4.2 Worm Selection for Injections
				3.4.3 Preparing Needles for Injections
				3.4.4 Injecting Worms
				3.4.5 Isolating MosSCI Transgenic Animals
			3.5 Synchronization of C. elegans Using Bleaching
			3.6 Aging of C. elegans for Age-Associated Studies
				3.6.1 Manual Removal of Animals from Progeny with a Standard  Pick
				3.6.2 Isolation of Adult Animals Using Washing and Sedimentation
				3.6.3 Chemical Sterilization of Adult Animals
			3.7 Visualization of the Actin Cytoskeleton in C. elegans
				3.7.1 Preparation of Slides for Imaging
				3.7.2 Considerations for Wide-Field/Compound Microscopy
			3.8 Quantitative Analyses of Actin Cytoskeletal Integrity in Worms
				3.8.1 Quantification of Actin Thickness in Muscle Filaments Using ImageJ
				3.8.2 Quantification of Actin Quantity in the Hypodermis Using a Large Particle Biosorter
			3.9 Physiological Measurements of C. elegans Health
				3.9.1 Life Span Measurements in C. elegans
				3.9.2 Thermotolerance in C. elegans
		4 Notes
		References
	Chapter 6: Imaging the Cytoskeleton in Living Plant Roots
		1 Introduction
		2 Materials
			2.1 M. truncatula Seedling Preparation
			2.2 Agrobacteria Preparation
			2.3 Hairy Root Transformation
			2.4 Live Cell Imaging of Roots and Agar Slabs for Root Mounting
		3 Methods
			3.1 Generation of M. truncatula Hairy Roots Expressing Fluorescent Cytoskeletal Reporters
				3.1.1 Preparing Seedlings
				3.1.2 Agrobacteria Preparation
				3.1.3 Hairy Root Transformation and Transgenic Root Screening
			3.2 Preparing A. thaliana Roots for Direct Live Cell Imaging
			3.3 Mounting Plant Roots for Microscopy Using Agar Slabs
		4 Notes
		References
	Chapter 7: Imaging of Actin Cytoskeleton in the Nematode Caenorhabditis elegans
		1 Introduction
			1.1 Immunofluorescent Staining of Actin
			1.2 Live Imaging of Actin Filaments
			1.3 Phalloidin Staining
		2 Materials
			2.1 C. elegans Culture
			2.2 Buffers
			2.3 Fixative
			2.4 Fluorescently Labeled Phalloidin
			2.5 Mounting
		3 Methods
			3.1 C. elegans Culture
			3.2 Harvesting and Fixing Worms for Phalloidin Staining
			3.3 Staining with TMR-Phalloidin
			3.4 Observation and Interpretation
		4 Notes
		References
	Chapter 8: Imaging of the Cytoskeleton Using Live and Fixed Drosophila Tissue Culture Cells
		1 Introduction
		2 Materials
			2.1 Culturing and Transient Transfection of Drosophila S2 Cells
			2.2 Fixation and Staining of Drosophila S2 Cells for Immunofluorescence
			2.3 Live-Cell Imaging
			2.4 Fixation and Preparation of Drosophila S2 Cells for Scanning Electron Microscopy
		3 Methods
			3.1 Culture of Drosophila S2 Cells (See Note 1)
			3.2 Transient Transfection of Drosophila S2 Cells (See Notes 2 and 3)
			3.3 Fixation and Staining of Drosophila S2 Cells for Immunofluorescence (See Note 7)
				3.3.1 Preparing the Surface of Coverslips (See Notes 8 and 9)
				3.3.2 Con A Coating of Coverslips
				3.3.3 Con A Coating of 96-Well Plates
				3.3.4 Polylysine Coating of Coverslips
				3.3.5 Coating of Coverslip-Bottom Imaging Chamber
				3.3.6 Plating of Cells (See Note 10)
				3.3.7 Fixation and Staining (See Note 11)
			3.4 Live-Cell Imaging (See Note 13)
			3.5 Fixation and Preparation of Drosophila S2 Cells for Scanning Electron Microscopy
				3.5.1 Fixation (See Note 15)
				3.5.2 Dehydration and Critical Point Drying (See Note 16)
				3.5.3 Mounting and Sputter Coating (See Note 17)
		4 Notes
		References
Part II: Cytoskeleton Dynamics
	Chapter 9: Influencing the Actin Dynamics in Plant Cells by Jasplakinolide, Chondramides, Phalloidin, Cytochalasins, and Latru...
		1 Introduction
			1.1 F-Actin-Stabilizing and F-Actin-Polymerizing Substances
				1.1.1 Jasplakinolide
				1.1.2 Chondramides
			1.2 Actin-Depolymerizing Substances
				1.2.1 Cytochalasins
				1.2.2 Latrunculins
				1.2.3 Swinholide
			1.3 Visualization of Inhibitory Effects
		2 Materials
			2.1 Cell Cultures
			2.2 Chemicals, Buffers, Solutions
				2.2.1 Actin-Perturbing Drugs and Solvents
				2.2.2 Electron Microscopy
				2.2.3 Immunoelectron Microscopy
				2.2.4 Fluorescence Microscopy
		3 Methods
			3.1 Treatment and Recovery Experiments
			3.2 Visualization of F-Actin After Drug Treatment
				3.2.1 Chemical Fixation/Electron Microscopy of Algal Cells after Holzinger and Meindl. The Procedure Described Herein Has Firs...
				3.2.2 Immunoelectron Microscopy
				3.2.3 Immunofluorescence Microscopy of Latrunculin B-Treated Maize Roots After Baluska et al.
				3.2.4 Immunofluorescence Microscopy of Latrunculin B-Treated Chlamydomonas Cells After Craig et al. and Craig and Avasthi
				3.2.5 Perfusion of Nitella Internodal Cells for FITC-Phalloidin Staining of F-Actin After Foissner and Wasteneys (Fig. 3; See ...
		4 Notes
		References
	Chapter 10: Analysis of Morphogenesis and Flagellar Assembly During Spermatogenesis in Planarian Flatworms
		1 Introduction
		2 Materials
			2.1 Planarians and Husbandry Medium
			2.2 Whole-Mount Sample Fixation and DAPI Staining
			2.3 Isolation of Testis Lobes and Labeling of Developing Sperm Nuclei and Flagella
			2.4 Visualization of Individual Sperm by Surgical Release from Vas Deferens
		3 Methods
			3.1 Handling and Maintenance of Planarian Colonies
			3.2 Whole-Mount Analysis of Testis Lobes by DAPI Staining
			3.3 Analysis of Spermatid Elongation and Flagellar Development in Isolated Planarian Testis Lobes
			3.4 Visualization of Individual Sperm by Surgical Release from Vas Deferens
		4 Notes
		References
	Chapter 11: An In Vitro Model System to Test Mechano-Microbiological Interactions Between Bacteria and Host Cells
		1 Introduction
		2 Materials
			2.1 Live Cell Imaging of F-Actin in Human Epithelial Cells
				2.1.1 Cell Culture and DNA Transfection
				2.1.2 Time-Lapse Fluorescence Microscopy
			2.2 Preparing Magnetic Beads Coated with Bacteria  Pili
				2.2.1 Purifying Pili from Neisseria gonorrhoeae
				2.2.2 Coating Beads with Purified  Pili
			2.3 Magnetic Tweezers Setup for Applying Local Force to Eukaryotic Cells
				2.3.1 Components of the Magnetic Tweezers
				2.3.2 Force Calibration
		3 Methods
			3.1 Preparations for Live Cell Imaging of F-Actin in Human Epithelial Cells
				3.1.1 Cell Culture and DNA Transfection
				3.1.2 Time-Lapse Fluorescence Microscopy
			3.2 Coating Magnetic Beads with Bacteria  Pili
				3.2.1 Purifying Pili from Neisseria gonorrhoeae
				3.2.2 Coating Beads with Purified  Pili
					Surface Activation of Beads with  EDC
					Immobilization of Ligand After Activation
					Quenching and Washing of Pili-Coated Beads
			3.3 Magnetic Tweezers Setup for Applying Local Force to a Eukaryotic  Cell
				3.3.1 Components of the Magnetic Tweezers
				3.3.2 Force Calibration
					Preparing a Mixture of Magnetic and Nonmagnetic Beads for Force Calibration
					Manipulating Tweezers for Force Calibration
					Qualitative In Situ Monitoring of the Magnetic Force
					Plotting ``Force vs. Distance´´ Curves
					Controlling the Amplitude and Speed of Force Pulling on Beads
				3.3.3 Tweezers Manipulation: Applying Force to Pili-Coated Beads
					Set Beads onto Epithelial Cells
					Position Tweezers over the Cells
					Time-Lapse Acquisition and ``Current vs. Time´´ Parameters
					Qualitative In Situ Monitoring of Pulling Forces
					Quantitative Analysis of Force-Dependent F-Actin Accumulation
		4 Notes
		References
	Chapter 12: Purification of Cytoskeletal Proteins by Fast Protein Liquid Chromatography (FPLC) Using an ÄKTA Start System
		1 Introduction
		2 Materials and Equipment
		3 Methods
			3.1 Prepare Reagents (See Notes 4 and 5)
			3.2 Engage the ÄKTA Start Instrument (See Note 4)
			3.3 Priming the  Pump
			3.4 Install/Change a Chromatography Column on the ÄKTA Start System (See Note 8)
			3.5 Equilibrate the Column
			3.6 Prime the Sample Tubing Using the  Pump
			3.7 Prime the Sample Loop Before Injecting Sample
			3.8 Write Methods in UNICORN Start (See Note 12)
			3.9 Equipment Shutdown and Short-Term Storage (See Note 13)
			3.10 Equipment Shutdown and Long-Term Storage (See Note 14)
		4 Notes
		References
	Chapter 13: The Cilioprotist Cytoskeleton, a Model for Understanding How Cell Architecture and Pattern Are Specified: Recent D...
		1 Introduction
		2 Basal Bodies
		3 Infraciliature
		4 Basal Body/Centriole Assembly
		5 De Novo Assembly of Basal Bodies
		6 Cilioprotist Cell Patterning and Architecture
		7 Pericentriolar Material (PCM)
		8 Nucleic Acids and Basal Bodies/Centrioles: DNA
		9 Nucleic Acids and Basal Bodies/Centrioles: RNA
		10 Origin of Centrioles and Basal Bodies
		11 Unanswered Questions and Unused Methods
			11.1 First of All, Why Use Cilioprotists as Models?
			11.2 Using Cilioprotists as Model Systems, What Do We Still Need to Learn?
				11.2.1 Nucleic Acids in C/BBs
					DNA
					RNA
				11.2.2 Proteins
				11.2.3 Nucleolini
			11.3 Newly Emerging Methods
		12 Final Thoughts
			12.1 Future Evolution of the Cytoskeleton in the Ciliated Protists
		References
Part III: Cell and Organelle Motility
	Chapter 14: 3D and 4D Tumorigenesis Model for the Quantitative Analysis of Cancer Cell Behavior and Screening for Anticancer D...
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 2D Coalescence Assay and mAb Screen
			2.3 3D Coalescence Assay for DIC Imaging
			2.4 3D Coalescence Overlay Assay with Cancer Cells
			2.5 3D Assay for Imaging with Laser Scanning Confocal Microscopy
		3 Methods
			3.1 Culturing Cells from Biopsy Tissue
			3.2 Preparation of 2D Coalescence Assay
			3.3 Preparation of 3D Coalescence Assay
			3.4 Preparation of 3D Coalescence Assay with Cancer Cells Overlaid onto Fibroblasts
			3.5 Preparation of 3D Assay for LSCM Imaging
				3.5.1 Labeling and Plating HUVECs
				3.5.2 Overlaying HUVECs with Cells in Matrigel
			3.6 2D and 3D Imaging and 3D Optical Sectioning
				3.6.1 2D Imaging
				3.6.2 3D Imaging and Optical Sectioning with DIC Microscopy
				3.6.3 LSCM
			3.7 Segmentation Using J3D-DIAS
			3.8 Reconstructions with J3D-DIAS
			3.9 J3D-DIAS 4.2 Quantitative Analysis of Cell Behavior
		4 Notes
		References
	Chapter 15: Melanosome Motility in Fish Retinal Pigment Epithelial (RPE) Cells
		1 Introduction
		2 Materials
			2.1 Poly-l-Lysine-Coated Coverslips
			2.2 Dissection and Isolation of RPE (Retinal Pigment Epithelial) Sheets
			2.3 Preparation of Dissociated RPE Cells
			2.4 Stimulation and Observation of Melanosome Motility
		3 Methods
			3.1 Preparation of Poly-l-Lysine-Coated Coverslips
			3.2 Dissection and Isolation of RPE Sheets
			3.3 Preparation of Dissociated RPE Cells
			3.4 Stimulation and Observation of Melanosome Motility
		4 Notes
		References
	Chapter 16: Chemotaxis: Under Agarose Assay
		1 Introduction
		2 Materials
			2.1 Agarose Plates
			2.2 Chemoattractants
			2.3 Developing Cells for cAMP Chemotaxis
			2.4 Time-Lapse Imaging
		3 Methods
			3.1 Folic Acid Chemotaxis: Preparation of the Agarose Plate
			3.2 Folic Acid Chemotaxis: Preparation and Loading of Amoebae and Chemoattractant
			3.3 cAMP Chemotaxis: Preparation of the Agarose Plate
			3.4 cAMP Chemotaxis: Preparation of cAMP-Responsive (Developing) Amoebae
			3.5 cAMP Chemotaxis: Loading the Cells and Chemoattractant into the Wells
			3.6 Imaging and Analysis
		4 Notes
		References
Part IV: Genetic and Proteomic Protocols
	Chapter 17: Functional Analysis of Actin-Binding Proteins in the Central Nervous System of Drosophila
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents
			2.3 Drosophila Strains and Culture (Available at the Bloomington Stock Center)
		3 Methods
			3.1 MARCM Analysis
				3.1.1 Preparation of FRT Dunc-115KG03651, Gal4, and Gal80 Strains
				3.1.2 Generation of MARCM Clones (See Note 5)
			3.2 Histology
				3.2.1 Larval Eye Disc/Brain Specimen Preparation
				3.2.2 Antibody Staining
		4 Notes
		References
	Chapter 18: Using a Hand-Held Gene Gun for Genetic Transformation of Tetrahymena thermophila
		1 Introduction
		2 Materials
			2.1 Growth and Starvation Media
			2.2 DNA Coating of Microcarriers
			2.3 Gene Gun
			2.4 Reagents for Selecting Transformants
		3 Methods
			3.1 Coating Vector DNA to Gold (AU) Microcarriers
			3.2 Coating the Plastic Tubing with Gold Microcarriers (See Note 8)
			3.3 Tube Cutting and Barrel Preparation
			3.4 Preparing Cells for Transformation Using Overexpression Vectors
			3.5 Preparing Conjugal Pairs for Biolistic Bombardment
			3.6 Using the Helios Hand-Held Gene Gun for Particle Bombardment
			3.7 Screening for Overexpression Transformants
			3.8 Somatic Transformations
			3.9 Screening for Somatic Transformants
		4 Notes
		References
	Chapter 19: Proteomic Tools for the Analysis of Cytoskeleton Proteins
		1 Introduction
		2 Materials
			2.1 Computer with Internet Access
			2.2 Visualization Software
				2.2.1 Visualization Software: Chimera
				2.2.2 Visualization Software: PyMOL
				2.2.3 Alternative Molecular Visualization Tools
				2.2.4 Modeling Software
		3 Methods
			3.1 Protein Sequences Databases
			3.2 Retrieval of Protein Sequences
				3.2.1 Retrieving Protein Sequences from NCBI´s GenPept
					Accession Number Search
					Keyword Search
				3.2.2 Retrieving Protein Sequences from UniProt
					Accession Number Search
					Keyword Search
			3.3 Text Formatting
			3.4 Searching for Similarities and Inferring Homologies
				3.4.1 Conducting a BLASTP (Protein-Specific BLAST) of the Nonredundant NCBI RefSeq Database
				3.4.2 Specialized BLAST Variants
				3.4.3 Conducting a PHMMER (Profile-HMM) Search
			3.5 Multiple Sequence Alignment
				3.5.1 Creation of a MSA Using CLUSTAL Omega
				3.5.2 Visualization of the MSA Using ESPript 3
			3.6 Protein Motifs, Patterns, and Domains
				3.6.1 InterPro
				3.6.2 PROSITE
				3.6.3 PFAM
				3.6.4 CDD
				3.6.5 SMART
				3.6.6 HHrepID
				3.6.7 REPRO
			3.7 Protein Secondary Structure Prediction
				3.7.1 PSIPRED
				3.7.2 PSSpred
				3.7.3 RaptorX-SS8
				3.7.4 SCRATCH
				3.7.5 JPred
			3.8 Prediction of Intrinsically Disordered Regions of Proteins
				3.8.1 DISOPRED3
				3.8.2 MFDp2
				3.8.3 PrDOS
			3.9 Retrieval of Three-Dimensional Coordinates of Known Protein Structures
			3.10 Modeling the Three-Dimensional Structure of a Protein or Domain Sequence
				3.10.1 Modeling Algorithms and Approaches
				3.10.2 Using HHpred to Generate a Homology Model Structure
				3.10.3 Using I-TASSER to Generate a Threading-Based Model
				3.10.4 Generating a Homology Model Using MODELLER
			3.11 Model Refinement
				3.11.1 ModRefiner
				3.11.2 SCWRL4
				3.11.3 3Drefine
				3.11.4 Refold
				3.11.5 refineD
			3.12 Model Evaluation Methods
				3.12.1 Verify 3D
				3.12.2 VoroMQA
				3.12.3 ProSA-web
				3.12.4 ProQ3
			3.13 Tools for Analyzing Structures
				3.13.1 Visualization of Protein Structure in Chimera
				3.13.2 Visualization of Protein Structure in PyMOL
				3.13.3 Manipulation of Protein Structures Using Chimera
			3.14 Prediction of Protein-Protein Interactions
				3.14.1 IntAct
				3.14.2 STRING
				3.14.3 BioGRID
			3.15 Docking Analysis
				3.15.1 AutoDock Vina
				3.15.2 LeDock
				3.15.3 ClusPro
		4 Notes
		References
Index