3ITN

Crystal structure of Pseudo-activated Procaspase-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
Collection date	2006-10-13
Detector	MARMOSAIC 300 mm CCD
Wavelength(s)	1.00
Spacegroup name	I 2 2 2
Unit cell lengths	69.224, 84.607, 96.171
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	33.790 - 1.630
R-factor	0.1987
Rwork	0.199
R-free	0.22420
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	Caspase-3 PDB code 2J30
RMSD bond length	0.005
RMSD bond angle	1.269
Data reduction software	DENZO
Data scaling software	SCALEPACK
Phasing software	CNS
Refinement software	CNS

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	33.790
High resolution limit [Å]	1.630	1.630
Rmerge	0.520
Number of reflections	32677
Completeness [%]	91.8	99
Redundancy	5.45

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	8.5	291.15	Proteins were dialyzed in a buffer of 10 mM Tris-HCl (pH 8.5) and 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 a 5:1 inhibitor:peptide ratio (w/w). 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 consisted of 10 mM Tris-HCl (pH 8.5), 10 mM DTT, and 3 mM NaN3. Crystals were obtained by the hanging drop vapor diffusion method. Concentrated protein (2 L) was mixed 1:1 with a reservoir solution that contained 100 mM sodium citrate (pH 5), 3 mM NaN3, 10 mM DTT, and 17% PEG 6000 (w/v). The crystallization plates were incubated at 18 C., VAPOR DIFFUSION, HANGING DROP, temperature 291.15K