دانلود کتاب Structural Proteomics: High-Throughput Methods

فهرست مطالب :

1. Introduction......Page 27
1.1. Target Selection......Page 28
1.2. Chapter Overview......Page 29
2.1.1. Overview of Fold Space......Page 30
2.1.2. Family Space......Page 32
2.1.3. Comparative Modeling......Page 35
2.1.4. Phylogenetic Distribution......Page 36
2.1.5. Assessing Similarity to Proteins of Known Structure......Page 37
2.2. Single-Genome Approach......Page 38
3. Target Selection:Addressing Limitations of High-Throughput Structure Determination......Page 39
3.1. Coiled Coils......Page 40
3.2. Transmembrane Proteins......Page 41
3.4.2. Disorder......Page 42
3.5. Additional Sequence Properties......Page 43
3.6. Domain Boundaries......Page 44
4. Target Selection: Future Approaches......Page 45
References......Page 47
C:\\Documents and Settings\\molbio\\Asztal\\EBL\\prot_9781588298096\\back-matter.pdf......Page 0
1. Introduction......Page 50
2.1.2. Ranking of Targets......Page 51
2.1.3. Manual Filtering......Page 52
2.2.1. Full-Length Protein or Protein Constructs?......Page 53
2.2.2. Selecting the Appropriate Criteria......Page 54
3. Notes......Page 55
References......Page 56
1.1.1. Tuberculosis......Page 59
1.2. Comparative Genomics......Page 60
1.3.1. Connectivity......Page 61
1.3.3. Disorder......Page 62
1.3.6. Gene Overlap......Page 63
1.5. Data Management......Page 64
3.1. Tuberculosis......Page 65
3.2. Sleeping Sickness......Page 66
References......Page 67
1. Introduction......Page 70
2.1. Limitations of Laboratory Notebooks and Spreadsheets......Page 72
2.2. Design Features of a LIMS for Structural Genomics......Page 73
2.4. The LIMS as a Data Mining Tool......Page 74
3.1.1. Introduction and Overview......Page 75
3.1.2.2. Crystallization......Page 76
3.1.2.3. Data Collection and Structure Determination......Page 78
3.2.1.1. Overall Goals......Page 79
3.2.1.6. Lab Resources......Page 80
3.2.1.10. Sesame Reliability......Page 81
3.2.2. Sesame Modules and Views......Page 82
3.2.3. Implementation......Page 87
3.2.3.1. Sesame Framework......Page 88
3.3.1. The Protein Production Data Model......Page 90
3.3.3.1. Experimental Workflow Management......Page 93
3.3.3.3. Searching Through Data and Generating Reports......Page 96
4. Conclusion......Page 97
References......Page 98
1. Introduction......Page 101
2.1. Data Representation......Page 102
2.1.2. mmCIF and the PDB Exchange Dictionary......Page 103
2.1.3. PDBML File Format......Page 104
2.2. Tools for Data Deposition and Annotation......Page 105
2.3. Tools for Tracking Progress of Structural Genomics Projects......Page 107
3.1. Data Deposition and Annotation......Page 108
3.1.1. Data Harvesting......Page 109
3.1.3.1. Checking the Identity of Chemical Components in the Deposition......Page 110
3.1.3.2. Checking the Sequence, Source, and Sequence Database......Page 111
3.1.3.3. Checking the Molecular Assembly......Page 112
3.1.4. Validation......Page 114
3.1.5. Interaction Between Depositors and Annotators......Page 115
3.2.3. Theoretical Models......Page 116
4. Future of Data Deposition and Annotation......Page 117
References......Page 118
1. Introduction......Page 122
1.2. Disorder Predictors......Page 123
3. Methods......Page 126
3.1.1. Prediction by Charge-Hydropathy Analysis......Page 127
3.1.2. Prediction by Position-Specific Scoring Schemes......Page 128
3.2. Predicting Exact Ordered Segments in a Protein......Page 129
4. Notes......Page 130
References......Page 132
1.1. How and When the “Domain Hypothesis” Emerged......Page 135
1.2. Domain Definition......Page 136
2.1. Experimental Methods......Page 137
2.2. Methods That Use Three-Dimensional Structure......Page 139
2.4. Methods Based on Similarity Search......Page 141
2.5. Methods Based on Multiple Sequence Alignments (MSAs)......Page 143
3.1. Databases Based on Sequence Analysis......Page 144
3.2. Databases Based on Three-Dimensional Information......Page 145
4. Domain Prediction-Lessons and Considerations......Page 146
4.1. Multiple Sequence Alignment as a Source for Domain Termini Signals......Page 148
4.2. Detecting Signals of Domain Termination......Page 149
4.3. Selecting the Most Likely Domain Partition......Page 151
4.3.1. Computing the Prior P(D)......Page 152
4.3.3. Generalization of the Likelihood Model to Multiple Proteins......Page 153
4.3.4. Selecting Representative Sequences......Page 154
5. Conclusions......Page 155
References......Page 156
1. Introduction......Page 162
2.4. Conventions Followed in the Text......Page 164
3.1. Fold Assignment......Page 165
3.3. Model Building......Page 166
4. Notes......Page 167
References......Page 174
1. Introduction......Page 177
2.2. Preparation of Inserts......Page 180
2.6. Clone Sequence Verification......Page 181
3.2. Preparation of Inserts......Page 182
3.4. Ligation and Transformation......Page 183
3.5. Colony PCR......Page 184
4. Notes......Page 185
References......Page 186
1. Introduction......Page 188
2.2.3. Liquid Culture Inoculation......Page 189
3.1. Competent Cells Preparation......Page 190
3.2. Automated Procedures......Page 191
3.2.2. Plating of Transformation Reactions......Page 194
3.3.2. SDS-PAGE......Page 195
4. Notes......Page 196
References......Page 199
1. Introduction......Page 200
2.2. Plasmids, Nucleotides, Gels, and Kits......Page 201
3.2. Amplification of Library Plasmid......Page 202
3.3. Screening of the Clones on the Basis of Gfp Fluorescence......Page 203
3.4. Test of Protein Expression as a Fusion with GFP......Page 204
4. Notes......Page 205
References......Page 208
1. Introduction......Page 209
2.2.1. Expression Vectors with Fusion Partners......Page 210
2.2.2. Expression Vectors for Protein Co-expression......Page 211
2.5. Protein Analysis......Page 212
2.6.2. Kits, Reagents, and Miscellaneous......Page 213
3.1.2. Cloning into Duet Vectors......Page 214
3.2.2.1. Expression in 96-Deep-Well Format......Page 215
3.2.2.2. HTP Screening for Protein Expression Using His-Select iLAPPlates:......Page 216
4. Notes......Page 218
References......Page 220
1. Introduction......Page 221
2.1. Apparatus for the High Throughput Pipeline......Page 222
2.2. PCR and PCR Results Test......Page 223
2.5. Cell Lysis and Protein Purification......Page 224
3.1. PCR and PCR Results Test......Page 225
3.2. BP Reaction......Page 226
3.3. Generating Entry Clones......Page 227
3.6.2. Preparing samples for protein purification......Page 228
3.7.2. ELISA......Page 229
4. Notes......Page 230
References......Page 232
1. Introduction......Page 233
2.1. Enzymes......Page 234
2.2. Bacteria and Plasmids......Page 235
2.6. Instruments......Page 236
2.8. Cloning......Page 237
3.1.1. Vector......Page 238
3.2.1. 96-Well PCR......Page 239
3.2.2. Vector Preparation......Page 240
3.2.4. Annealing and Transformation......Page 242
3.2.5. Colony Screening......Page 243
3.3.2. Cell Growth and Induction......Page 244
3.3.3. Small-Scale Purification and Analysis......Page 245
3.4.1. Culture Growth and Induction......Page 246
3.4.3. Cell Extraction......Page 247
3.4.5. Manual Affinity Purification......Page 248
3.4.6. Analysis, Further Purification, and Storage......Page 249
4. Notes......Page 251
References......Page 257
1. Introduction......Page 259
2.1. Several Vectors......Page 260
2.3. Single Vector-Multiple Transcripts Strategies......Page 261
4. Coexpression in Mammalian Cells......Page 262
5. Flowchart of High Throughput Protein Complexes Assembly by Coexpression......Page 264
6. Notes......Page 265
References......Page 267
1. Introduction......Page 269
2. Materials......Page 270
3.1. Preparation of the S30 Extract......Page 271
3.1.4. Day Four: Preparation of the S30 Extract......Page 272
3.2. Stock Solutions for Cell-Free Protein Production......Page 273
3.3. Preparation of the Amino-Acid Stock Solutions......Page 274
3.4.1. Preparation of the Reaction Chamber......Page 275
3.4.2. Preparation of the Reaction Mixtures......Page 276
3.4.2.3. Preparation of the Reaction Mixture and Outside Buffer......Page 277
3.5. Preparation of the Samples for NMR Analysis......Page 278
References......Page 279
1. Introduction......Page 281
2.2. High Throughput, Automated Refolding Assay......Page 282
3.1.2. Refolding and Elution......Page 283
3.2.2. Analysis of Refolding Yield......Page 284
4. Notes......Page 285
References......Page 286
1. Introduction......Page 288
1.1. Practical Observations Regarding Membrane Protein Production......Page 289
2.1. Target Identification and Cloning......Page 293
2.2. Cell Growth, Lysis and Expression Analysis......Page 294
3.1. Target Identification and Cloning......Page 295
3.2. Cell Growth, Lysis, and Expression Analysis......Page 296
3.3.2. Nickel Affinity Chromatography......Page 297
3.3.3. TEV Protease Digestion......Page 298
3.4. Crystal Trials......Page 299
3.6. Conclusion......Page 300
4. Notes......Page 301
References......Page 305
1. Introduction......Page 307
1.3. Protein Characterization by Static Light Scattering......Page 308
2.1.4. Preparation and Automated Mass Spectrometry of Peptide Samples......Page 309
2.3. Static Light Scattering......Page 310
3.1.1. Preparation and Automated Mass Spectrometry of Intact Protein Samples......Page 311
3.1.2. Preparation of N-Glycosylated Samples......Page 312
3.1.3. Preparation of Samples from Crystals (11)......Page 313
3.1.5. Preparation of Samples for N-Glycosylation Site Analysis and Interpretation of Results......Page 314
3.2. ThermoFluor Assay......Page 316
3.3.1. Experimental Set-up and Calibration......Page 318
3.3.3. Data Analysis......Page 320
4. Notes......Page 321
References......Page 326
1. Introduction......Page 327
2.1. Multi-Well Plate Method......Page 328
3.1. Multi-Well Plate Method......Page 329
3.1.1. Analysis of Standards for Multi-Well Plate Method......Page 330
3.2.1. Preparation of Standards and Samples for HPLC Assay......Page 331
3.2.2. HPLC Assay......Page 332
4. Notes......Page 333
References......Page 337
1. Introduction......Page 339
2.2. General Screens......Page 340
2.3. Natural Phosphatase......Page 342
3.1. General Screens......Page 343
3.2. Phosphatase Screen Using Natural Substrates......Page 344
3.4. Nucleotide Pyrophosphatase......Page 345
4. Notes......Page 347
References......Page 348
1. Introduction......Page 350
1.1. Orthologue Screening......Page 353
1.3. Deletion Construct Generation......Page 354
2.3. Deletion Construct Generation......Page 355
3.2.2. NMR Spectroscopy......Page 356
3.3.2. Example: B. halodurans hypothetical protein BH2331 (10174951)......Page 358
4.1. Orthologue Screening......Page 360
4.2. 1D 1H NMR Fold Screening......Page 361
4.3. Deletion Construct Generation......Page 362
5.3. Deletion Construct Generation......Page 363
6. Summary......Page 364
References......Page 365
1. Introduction......Page 368
2.2. Microcapillary Crystallization in Nanoliter Volume......Page 370
3.1. Microcapillary Protein Crystallization for Initial Screening......Page 371
3.2. Counter-Diffusion Crystallization......Page 372
3.3. Visualization and X-Ray Analysis......Page 373
3.4. Preparation of Heavy Atom Soaking and Cryogenic Preservation......Page 376
3.5. Synchrotron Radiation......Page 377
4. Notes......Page 378
References......Page 379
1. Introduction......Page 382
2.2. Protein Crystallization Screening......Page 383
3.1. Preparation of CR-Derivatized Protein......Page 384
3.3. Fluorescent Observations......Page 385
3.4. Bright Fluorescent Regions as Potential Lead Conditions......Page 386
4. Notes......Page 389
References......Page 390
1. Introduction......Page 392
2.1. Crystallization Screens......Page 393
2.4. Drop-Based Crystallization......Page 394
3.1. Experimental Strategy......Page 395
3.2.1.2. High Purity......Page 396
3.3.1. SDS-PAGE......Page 397
3.4. Setting up a Microfluidic Crystallization Experiment......Page 398
3.5. Interpreting the Results of a Microfluidic Crystallization Experiment......Page 399
3.5.2. Crystals......Page 400
3.6.1. Translation Using the Original Reagent (Microbatch)......Page 401
4. Notes......Page 402
References......Page 406
1. Introduction......Page 408
2. Materials......Page 411
4. Notes......Page 412
References......Page 413
1. Introduction......Page 415
3.2. Optimization of the Hit Growth Solution......Page 417
References......Page 422
1. Introduction......Page 423
2.1. User Interfaces......Page 424
2.1.3. Wizards......Page 425
2.2. Common Crystallographic Computations......Page 426
2.2.2. Automated Structure Solution via Molecular Replacement......Page 427
2.2.4. Refinement......Page 428
2.2.6. Twinned Data......Page 429
3.2. Molecular Replacement......Page 430
3.4. Refinement......Page 432
3.5. Twinning......Page 434
4. Notes......Page 435
References......Page 437
1. Introduction......Page 440
2.1. M9 Medium for Uniform 15N-Labeling......Page 441
3.1. Uniform 15N-Labeling in M9 Medium......Page 442
3.2. Uniform 15N-Labeling in Autoinduction Medium......Page 443
3.3. NMR Sample Preparation......Page 444
3.4. NMR Screening......Page 445
4. Notes......Page 446
References......Page 448
1. Introduction......Page 450
2.1. NMR Microcoil Probes......Page 453
2.2. Cleaning Is Critical for the Long-Term Performance of Flow-Through Microcoil NMR Probes......Page 454
3. Methods......Page 455
3.1. Miniaturized NMR Screening Using Microcoil NMR Probes......Page 456
3.2. Microcoil NMR Probes Enable the Design of Novel Pulse-Sequences:A Novel Aliphatic-Aromatic HCCH-TOCSY Experiment......Page 457
3.3. Three-Dimensional Structure Determination Using Microcoil NMR Probes......Page 458
References......Page 459
1. Introduction......Page 462
1.2.1. Amine-Reactive Cross-linking Reagents (NHS Esters, Imidoesters)......Page 463
1.2.2. Maleimides (Sulfhydryl-Reactive Reagents)......Page 464
1.2.3. Photoreactive Reagents......Page 465
1.4. Identification of Cross-links......Page 466
1.4.4. MALDI-TOF/TOF-MS of Cleavable Cross-linkers......Page 467
1.5.1. MALDI-TOF Mass Spectrometry......Page 468
1.7. General Methodology......Page 469
2.1. Chemical Cross-linking......Page 470
3. Methods......Page 471
3.3. MALDI Mass Spectrometry......Page 472
3.6. Docking with Distance Constraints......Page 473
4. Notes......Page 475
References......Page 476
1.2. Objective......Page 478
1.3. Pipeline......Page 479
3.1. Method......Page 480
3.2. Databases......Page 481
3.4. Identifying MP Targets Predicted To Be Less Tractable for High Throughput Study......Page 482
3.7. Target Deselection......Page 483
4.3. Large-Scale Preparation of Cell Paste......Page 484
4.6. Protein Production Summary......Page 485
5.2. On-Column Refolding......Page 486
6.1. Crystallization......Page 487
7. Summary of BSGC Throughput during the PSI-1 Pilot Phase......Page 489
8.2. Mapping of the Protein Structure Space......Page 491
8.4. Structural Families Found in the Minimal Organism......Page 493
References......Page 494
1. Introduction......Page 498
2. Pipeline Overview......Page 499
2.1. Target Selection......Page 500
2.2. Protein Production......Page 502
2.3. Protein Crystallization......Page 503
2.4. Data Collection, Processing, and Structure Determination......Page 504
3. Technology Development......Page 505
3.1. Computational Domain Prediction and Chunk Selection......Page 506
3.2. Design, Synthesis, and Systematic Testing of Co-Crystallants......Page 507
3.3. Capillary Crystal-Growth Robotics......Page 508
4. Medical Structural Genomics......Page 510
References......Page 511
1. Introduction......Page 515
2.3. Diffraction Screening......Page 517
3. Notes......Page 518
References......Page 519
1. Introduction......Page 522
2.2.3. Gateway Cloning......Page 524
3.1. Target Selection......Page 525
3.2.1. Primer Design......Page 526
3.2.3. Gateway Cloning......Page 527
3.3. Protein Production......Page 528
3.4.2. Crystallization Set-up Using the Tecan Genesis Workstation Robot......Page 529
3.4.4. Crystallization Plate Visualization......Page 530
4. Conclusion......Page 531
5. Notes......Page 532
References......Page 533
1. Introduction......Page 536
2.2. Small-Scale Protein Expression and Purification Testing......Page 537
2.3.4. GST Fusion Elution and TEV Cleavage in Solution......Page 538
3.1. Target Selection......Page 539
3.3. Cloning Procedures......Page 540
3.4. Small-Scale Protein Expression and Purification Testing......Page 542
3.5.2.1. Ni-NTA Affinity Purification, No Cleavage......Page 545
3.6. Quality Control of Purified Proteins......Page 546
3.7.1. Protocol for Limited Proteolysis......Page 548
3.8. Protein Crystallization Screening......Page 549
3.9. Structure Determination......Page 550
3.10. Examples of Protein Structures and Their Complexeswith Ligands......Page 551
4. Notes......Page 553
References......Page 555
1. Introduction......Page 559
3.1.1. Targets and Primers......Page 560
3.1.2. PCR......Page 561
3.1.4. Picking and Screening Cloned Colonies......Page 562
3.1.7. Expression and Solubility Testing......Page 563
3.2.2. Large-Scale Fermentation of Selenomethionine Labeled Proteins......Page 564
3.2.3. Large-Scale Fermentation of Native Proteins......Page 565
3.3.4. Protein Quality Control......Page 566
3.4. SGX Crystallization......Page 567
3.4.2. Crystal Optimization......Page 568
3.4.4. Crystallization and LIMS......Page 569
4. Notes......Page 570
References......Page 573
1. Introduction......Page 574
2.1. Target Selection......Page 575
2.3. Expression and Purification......Page 577
2.4. Protein Refolding......Page 578
2.7.1. Protein Concentration Optimization......Page 579
2.9. Functional Characterization......Page 580
3.2. Functions Revealed......Page 581
References......Page 582
1. Introduction......Page 585
3.1. Target Selection......Page 586
3.3. Protein Expression and Solubility Studies......Page 587
3.6. Protein Crystallography......Page 588
4.2. Functions Revealed......Page 589
References......Page 590
Kobe_Index.pdf......Page 592