7JL7

Zebrafish Caspase N213T

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	2018-08-18
Detector	MARMOSAIC 300 mm CCD
Wavelength(s)	1.0
Spacegroup name	P 21 21 21
Unit cell lengths	56.305, 74.580, 133.413
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	35.910 - 2.050
R-factor	0.2051
Rwork	0.202
R-free	0.25330
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	5jft
Data reduction software	HKL-2000
Data scaling software	HKL-2000
Phasing software	PHENIX
Refinement software	PHENIX (1.18.2_3874)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	35.910	2.110
High resolution limit [Å]	2.050	2.070
Number of reflections	32428	91
<I/σ(I)>	30.77
Completeness [%]	90.5
Redundancy	5.1
CC(1/2)	0.843	0.069

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP		291	Protein was dialyzed in a buffer of 10 mM Tris-HCl, pH 8.5, 1 mM DTT, concentrated to 4 mg/mL, and inhibitor Ac-DEVD-CHO was added at a 5:1 (w/w) inhibitor/protein ratio. Crystals were obtained at 18 C by the hanging drop vapor diffusion method using a 4 uL drop that contained equal amounts of protein and reservoir solution. Each well contained a reservoir solution (500 uL) of 100 mM sodium citrate, pH 5.4, 23% PEG 6000, 10 mM DTT, and 3 mM NaN3. Flat, sheet-like crystals appeared within 14 days, and we used microseeding to obtain diffraction-quality crystals. In this case, crystal trays were set up as described above and incubated for 24 hours. The flat sheet crystals were collected and treated with seed beads using a kit from Hampton Research. A 4 uL drop containing flat sheet crystals was added to a tube containing seed beads. Reservoir solution (10 uL) was pipetted on the cover slide to remove all crystals from the coverslip, and the procedure was repeated five times. The resulting mixture of crystals and seed beads was vortexed for 30 seconds and cooled on ice for 10 seconds, and the procedure was repeated six times. Serial dilutions of the treated crystals were set up from 10-1 to 10-3, and 0.5 uL of the 10-2 dilution crystal seeds was added into the drops of the 24-hour crystal tray. Larger cube-shaped crystals appeared within 14 days. The crystals were collected and frozen in liquid nitrogen following the addition of 20% MPD plus the reservoir solution.