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 |