2IAG

Crystal structure of human prostacyclin synthase

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	NSRRC BEAMLINE BL13B1
Synchrotron site	NSRRC
Beamline	BL13B1
Temperature [K]	100
Detector technology	CCD
Collection date	2006-06-29
Detector	ADSC QUANTUM 315
Wavelength(s)	0.980127
Spacegroup name	P 1 21 1
Unit cell lengths	67.733, 105.596, 73.950
Unit cell angles	90.00, 92.01, 90.00

Refinement procedure

Resolution	20.150 - 2.150
R-factor	0.20407
Rwork	0.201
R-free	0.26313
Structure solution method	MAD
RMSD bond length	0.011
RMSD bond angle	1.380
Data reduction software	HKL-2000
Data scaling software	HKL-2000
Phasing software	SOLVE
Refinement software	REFMAC (5.2.0019)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	30.000	2.230
High resolution limit [Å]	2.150	2.150
Number of reflections	51322
<I/σ(I)>	12.7	3.1
Completeness [%]	90.9	93.5
Redundancy	2.8

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	8	277	The native recombinant PGIS (25 mg/ml in the buffer containing 20 mM Tris, pH 8.0 and 150 mM NaCl) was found to crystallize spontaneously into needle clusters in the Eppendorf tube when stored at 4 C for about a week. A combination of microseeding technique and the hanging-drop vapor-diffusion method was then used to improve crystal quality. Specifically, seed stock was prepared by crushing these initial PGIS needle clusters with the Seed Bead Kit (Hampton Research). Following four 10-fold serial dilutions of the seed stock using the gel filtration buffer, 1 micro-l of diluted seeds was added into a 10 micro-l drop of freshly concentrated PGIS sample, and equilibrated at 4 C against 450 micro-l of the gel filtration buffer. High quality single crystals of PGIS were most often obtained using 100- or 1000-fold diluted seed stock, and reached suitable size (~ 0.05,0.05 ~ 0.1 mm3) within one week., VAPOR DIFFUSION, HANGING DROP, temperature 277K
1	VAPOR DIFFUSION, HANGING DROP	8	277	The native recombinant PGIS (25 mg/ml in the buffer containing 20 mM Tris, pH 8.0 and 150 mM NaCl) was found to crystallize spontaneously into needle clusters in the Eppendorf tube when stored at 4 C for about a week. A combination of microseeding technique and the hanging-drop vapor-diffusion method was then used to improve crystal quality. Specifically, seed stock was prepared by crushing these initial PGIS needle clusters with the Seed Bead Kit (Hampton Research). Following four 10-fold serial dilutions of the seed stock using the gel filtration buffer, 1 micro-l of diluted seeds was added into a 10 micro-l drop of freshly concentrated PGIS sample, and equilibrated at 4 C against 450 micro-l of the gel filtration buffer. High quality single crystals of PGIS were most often obtained using 100- or 1000-fold diluted seed stock, and reached suitable size (~ 0.05,0.05 ~ 0.1 mm3) within one week., VAPOR DIFFUSION, HANGING DROP, temperature 277K