1V11
CROSSTALK BETWEEN COFACTOR BINDING AND THE PHOSPHORYLATION LOOP CONFORMATION IN THE BCKD MACHINE
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | SYNCHROTRON |
Source details | APS BEAMLINE 19-ID |
Synchrotron site | APS |
Beamline | 19-ID |
Temperature [K] | 100 |
Detector technology | CCD |
Collection date | 2003-07-25 |
Spacegroup name | P 31 2 1 |
Unit cell lengths | 145.505, 145.505, 69.172 |
Unit cell angles | 90.00, 90.00, 120.00 |
Refinement procedure
Resolution | 27.520 - 1.950 |
R-factor | 0.139 |
Rwork | 0.138 |
R-free | 0.17100 |
Structure solution method | MOLECULAR REPLACEMENT |
Starting model (for MR) | 1ols |
RMSD bond length | 0.020 |
RMSD bond angle | 1.714 |
Data reduction software | HKL-2000 |
Data scaling software | HKL-2000 |
Refinement software | REFMAC (5.1.24) |
Data quality characteristics
Overall | Outer shell | |
Low resolution limit [Å] | 33.350 | 1.980 |
High resolution limit [Å] | 1.950 | 1.950 |
Rmerge | 0.088 | 0.606 |
Number of reflections | 61649 | |
<I/σ(I)> | 19 | 2.6 |
Completeness [%] | 100.0 | 100 |
Redundancy | 6 | 6 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION | 5.5 | 293 | CRYSTALS WERE GROWN AT 20C VIA THE VAPOR DIFFUSION METHOD BY MIXING EQUAL AMOUNTS OF PROTEIN (20-25 MG/ML IN 50 MM HEPES/NAOH, PH 7.5, 250 MM KCL, 0.5 MM PMSF, 1 MM BENZAMIDINE AND 5% (V/V) GLYCEROL) WITH WELL SOLUTION (1.4-1.6 M AMMONIUM SULFATE, 0.1 M NA-CITRATE PH 5.8, 20 MM B-MERCAPTOETHANOL). SERIALLY DILUTED CRUSHED CRYSTALS WERE USED FOR MICRO-SEEDING ONE DAY AFTER THE DROPS WERE SET UP. CRYSTALS APPEARED ONE DAY AFTER SEEDING AND GREW TO A MAXIMUM SIZE OF 120 X 800 UM WITHIN 10 DAYS. CRYSTALS WERE STABILIZED FOR 12 HOURS BY TRANSFER TO FRESH WELL SOLUTION. THEY WERE THEN CRYO-PROTECTED BY STEP-WISE TRANSFER INTO CRYO-BUFFER CONTAINING 1.6 M AMMONIUM SULFATE, 50 MM HEPES, PH 7.5, 100 MM NA-CITRATE, PH 5.8, 100 MM KCL, 50 MM DTT AND UP TO 20% (V/V) GLYCEROL. IT WAS FOUND THAT MN2+ IONS COULD REPLACE THE MG2+ REQUIRED FOR THE BINDING OF THDP TO THE ENZYME. |