5IAR
Caspase 3 V266W
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | SYNCHROTRON |
Source details | APS BEAMLINE 22-BM |
Synchrotron site | APS |
Beamline | 22-BM |
Temperature [K] | 100 |
Detector technology | CCD |
Collection date | 2012-10-11 |
Detector | MARMOSAIC 225 mm CCD |
Wavelength(s) | 1.00 |
Spacegroup name | I 2 2 2 |
Unit cell lengths | 68.871, 84.628, 96.237 |
Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
Resolution | 31.780 - 1.760 |
R-factor | 0.161 |
Rwork | 0.159 |
R-free | 0.18500 |
RMSD bond length | 0.007 |
RMSD bond angle | 0.926 |
Data reduction software | HKL-2000 |
Data scaling software | HKL-2000 |
Phasing software | PHENIX |
Refinement software | PHENIX ((1.10.1_2155: ???)) |
Data quality characteristics
Overall | |
Low resolution limit [Å] | 31.776 |
High resolution limit [Å] | 1.761 |
Number of reflections | 28121 |
<I/σ(I)> | 1.35 |
Completeness [%] | 99.7 |
Redundancy | 7.2 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, HANGING DROP | 8.5 | 291 | Caspase-3 variants were crystallized in the presence of Ac-DEVD-CMK. 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. |