2J33

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	67.376, 83.297, 95.915
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	33.230 - 2.000
R-factor	0.176
Rwork	0.176
R-free	0.20500
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.006
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	2.070
High resolution limit [Å]	2.000	2.000
Rmerge	0.080	0.260
Number of reflections	18581
<I/σ(I)>	31.7	7.7
Completeness [%]	98.5	97.2
Redundancy	6.9	6.5

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.