2KHO
NMR-RDC / XRAY structure of E. coli HSP70 (DNAK) chaperone (1-605) complexed with ADP and substrate
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | KAPPA-SHIFTED 15N-1H HSQC- TROSY | 0.2 MM [U-100% 13C U-100% 15N U-80% 2H] HSP70, 10 MM POTASSIUM CHLORIDE, 25 MM TRIS, 10 MM DTT, 5 MM MGCL2, 5 MM ADP, 10 MM POTASSIUM PHOSPHATE, 0.2 MM SODIUM AZIDE, 2 MM NRLLLTG, 90% H2O/ 10% D2O | 90% H2O/10% D2O | 0.05 | 7.2 | AMBIENT | 300 | |
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Varian | INOVA | 800 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| RDC OPTIMIZATION | RDC's WERE FITTED FOR (IA, IB, IIA) (IIB) (BETA, LID) AS SEPARATE UNITS USING GRID SEARCH OVER DA, DR, AND THREE EULER ANGLES FOLLOWED BY STEEPEST DESCEND. XRAY DATA FROM PDB ENTRY 1DKG WERE USED FOR RESIDUES 3-378 (NBD - NUCLEOTIDE BINDING DOMAIN). XRAY DATA FROM PDB ENTRY 1DKX WERE USED FOR 397-603 (SBD - SUBSTRATE BINDING DOMAIN). MISSING LOOPS IN 1DKG WERE ANNEALED AND MINIMIZED USING SWISSPROT SERVER. ORIENTATION OF DOMAIN IIB (RESIDUES 229-307) ADAPTED TO NMR DATA. DOMAIN IIB WAS ROTATED 20 DEGREES BASED ON RDC DATA AND MINIMALLY SUPERPOSED ON IIB IN 1DKG USING TRANSLATION AND ROTATION AROUND SZZ ONLY. CONNECTING RESIDUES 225-233 AND 307-314 WERE MINIMIZED IN SWISSPROT. NBD AND SBD WERE ORIENTED WITH RESPECT TO EACHOTHER BASED ON THE NMR DIPOLAR INFORMATION TRANSLATIONAL POSITION OF SBD WITH RESPECT TO NBD WAS DETERMINED BY COMPUTING THE BEST THEORETICAL ALLIGNMENT TENSOR USING PALES FROM ZWECKSTETTER M & BAX A (2001) J BIOMOL NMR 20(4):365-377. HETATM RECORDS IDENTIFY TENSOR ORIENTAIONS FOR DOMAINS IA,IB AND IIA AND BETA-LID OBTAINED FROM SELF_VALIDATION USING 50% OF THE RDC DATA. 65 A Z SEPARATION BETWEEN COORDINATE CENTERS OF NBD AND BETA-LID. 10 A Y SEPARATION BETWEEN COORDINATE CENTERS OF NBD AND BETA-LID. LINKER RESIDUES 379-395 ARE DYNAMIC AND WERE PLACED IN ARBITRARY CONFORMATION USING MOE (MOLECULAR OPERATING ENVIRONMENT) CHEMICAL COMPUTING GROUP 1010 SHERBROOKE ST. W, SUITE 910 MONTREAL, QUEBEC, CANADA H3A 2R7. LINKER CONFORMATION WAS OPTIMIZED IN MOE AND SWISS PROT. | REDCAT |
| NMR Ensemble Information | |
|---|---|
| Conformer Selection Criteria | target function |
| Conformers Calculated Total Number | 1 |
| Conformers Submitted Total Number | 1 |
| Representative Model | 1 (fewest violations) |
| Additional NMR Experimental Information | |
|---|---|
| Details | NMR 1H-15N RESIDUAL DIPOLAR COUPLING (RDC) DATA IN 4% STRETCHED POLY ACRYL AMIDE. NH RDC DATA WERE OBTAINED FROM KAPPA-SHIFTED TROSY USING A VARIAN INOVA 800 MHZ NMR SPECTROMETER EQUIPPED WITH A HCN COLD (CRYO) PROBE. THE NUCLEOTIDE-BINDING DOMAIN (NBD) AND SUBSTRATE BINDING DOMAIN (SBD) ARE CONNECTED BY A FLEXIBLE LINKER AND MOVE WITH RESPECT TO EACHOTHER IN A CONE WITH AN ESTIMATED OPENING ANGLE OF 70 DEGREES ON THE NANO SECOND TIME SCALE. |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | refinement | REDCAT | VALAFAR, H. AND PRESTEGARD, J.H.(2004) | |
| 2 | structure solution | REDCAT | VALAFAR, H. AND PRESTEGARD, J.H.(2004) | |
| 3 | refinement | own software FORTRAN 77 | E. ZUIDERWEG, 2008 | |
