5TJK

Crystal structure of GTA + A trisaccharide (native)

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	ROTATING ANODE
Source details	RIGAKU MICROMAX-002
Temperature [K]	113
Detector technology	IMAGE PLATE
Collection date	2009-04-16
Detector	RIGAKU RAXIS IV++
Wavelength(s)	1.5418
Spacegroup name	C 2 2 21
Unit cell lengths	52.550, 148.660, 79.790
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	19.690 - 1.450
R-factor	0.1825
Rwork	0.182
R-free	0.19270
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1lz0
RMSD bond length	0.010
RMSD bond angle	1.448
Data reduction software	d*TREK
Data scaling software	d*TREK (9.7 W8RSSI)
Phasing software	PHASER
Refinement software	REFMAC (5.8.0155)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	19.690	19.690	1.500
High resolution limit [Å]	1.450	3.120	1.450
Rmerge	0.036	0.021	0.310
Rmeas	0.036
Total number of observations	237616	27553	18945
Number of reflections	55231
<I/σ(I)>	17.7	53.7	3.6
Completeness [%]	99.1	98.1	97.8
Redundancy	4.27	4.77	3.5

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP		277	Native crystals of GTA/GTB lacking any heavy metals were grown at 4 degrees Celsius from much higher concentrations of protein (30-40 mg/mL for GTB and 16-20 mg/mL for GTA) along with 1% PEG 4000, 4.5-5% 2-methyl-2,4-pentanediol (MPD), 100 mM ammonium sulfate, 70 mM sodium chloride, 50 mM ADA buffer pH 7.5, 30 mM sodium acetate buffer pH 4.6 and 5 mM MnCl2 for GTB crystallization and 5-8 mM CoCl2 for GTA crystallization. 10-15 microlitre drops were placed against a reservoir containing 3.7% PEG 4000, 7% MPD, 0.3 M ammonium sulfate, 0.25 M sodium chloride, 0.2 M ADA buffer and 0.1 M sodium acetate. The crystals were usually grown for 5-10 days. Before making complexes, crystals of GTA/GTB were washed with modified mother liquor ML-2 consisting of 3.5% PEG 4000, 50 mM ammonium sulfate, 40 mM sodium chloride, 35 mM ADA buffer and 15% MPD or glycerol. Crystals of native GTA/GTB in complex with the respective A or B trisaccharide were obtained by soaking them in mother liquor ML-2 with 15% glycerol or MPD and 45-50 mM of each substrate for 2-5 days at 4 degrees Celsius. Before freezing the crystals for data collection, the concentration of the cryoprotectant was made 30% glycerol or 20% MPD respectively