2J30
The Role of Loop Bundle Hydrogen Bonds in the Maturation and Activity of (Pro)caspase-3
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | SYNCHROTRON |
Source details | APS BEAMLINE 22-ID |
Synchrotron site | APS |
Beamline | 22-ID |
Temperature [K] | 100 |
Detector technology | CCD |
Detector | MARRESEARCH |
Spacegroup name | I 2 2 2 |
Unit cell lengths | 68.732, 84.395, 96.352 |
Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
Resolution | 27.010 - 1.400 |
R-factor | 0.197 |
Rwork | 0.197 |
R-free | 0.20700 |
Structure solution method | MOLECULAR REPLACEMENT |
RMSD bond length | 0.005 |
RMSD bond angle | 1.300 |
Data reduction software | HKL-2000 |
Data scaling software | SCALEPACK |
Refinement software | CNS (1.1) |
Data quality characteristics
Overall | Outer shell | |
Low resolution limit [Å] | 50.000 | 1.450 |
High resolution limit [Å] | 1.400 | 1.400 |
Rmerge | 0.060 | 0.750 |
Number of reflections | 54279 | |
<I/σ(I)> | 46.2 | 1.35 |
Completeness [%] | 97.9 | 83.8 |
Redundancy | 5.8 | 3.5 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, HANGING DROP | 8.5 | 291 | PROTEINS WERE DIALYZED IN A BUFFER OF 10 MM TRIS-HCL, PH 8.5, 1 MM DTT. THE PROTEIN WAS CONCENTRATED TO 10 MG/ML USING AMICON ULTRAFREE CENTRIFUGAL FILTER DEVICES, AND INHIBITOR, AC-DEVD-CMK RECONSTITUTED IN DMSO, WAS THEN ADDED AT 5:1 WT:WT, INHIBITOR TO PEPTIDE. THE PROTEIN WAS DILUTED TO A CONCENTRATION OF 8 MG/ML BY ADDING 10 MM TRIS-HCL, PH 8.5, CONCENTRATED DTT AND CONCENTRATED NAN3 SO THAT THE FINAL BUFFER WAS 10 MM TRIS-HCL, PH 8.5, 10 MM DTT, 3 MM NAN3. 2 UL OF CONCENTRATED PROTEIN WAS MIXED 1:1 WITH WELL BUFFER THAT CONTAINED 100 MM SODIUM CITRATE, PH 5, 3 MM NAN3, 10 MM DTT AND 17% PEG 6000 W/V. SOLUTIONS WERE INCUBATED AT 18 DEG C USING THE HANGING DROP METHOD. CRYSTALS GREW WITHIN THREE DAYS FOR WILD- TYPE CASPASE-3 AND WITHIN TWO WEEKS FOR THE MUTANTS. THE IDEAL FREEZING CONDITIONS WERE FOUND TO BE WITH 80% MOTHER LIQUOR AND 20% PEG 400. |