5IBR

Caspase 3 V266K

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	2015-03-06
Detector	MARMOSAIC 225 mm CCD
Wavelength(s)	1.00
Spacegroup name	C 1 2 1
Unit cell lengths	108.968, 96.338, 68.704
Unit cell angles	90.00, 129.13, 90.00

Refinement procedure

Resolution	48.170 - 1.740
R-factor	0.182
Rwork	0.182
R-free	0.20200
RMSD bond length	0.007
RMSD bond angle	1.116
Data reduction software	HKL-2000
Data scaling software	HKL-2000
Phasing software	PHENIX
Refinement software	PHENIX (1.8.1_1168)

Data quality characteristics

	Overall
Low resolution limit [Å]	48.169
High resolution limit [Å]	1.738
Number of reflections	56481
<I/σ(I)>	1.39
Completeness [%]	99.9
Redundancy	3.8

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.