5T7E

Crystal structure of Streptomyces hygroscopicus Bialaphos Resistance (BAR) protein in complex with Coenzyme A and L-phosphinothricin

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 24-ID-C
Synchrotron site	APS
Beamline	24-ID-C
Temperature [K]	90
Detector technology	PIXEL
Collection date	2015-08-08
Detector	DECTRIS PILATUS 6M-F
Wavelength(s)	0.987
Spacegroup name	P 1 21 1
Unit cell lengths	64.180, 66.110, 86.150
Unit cell angles	90.00, 103.06, 90.00

Refinement procedure

Resolution	62.520 - 1.800
R-factor	0.2098
Rwork	0.208
R-free	0.24710
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.002
RMSD bond angle	0.590
Data reduction software	iMOSFLM
Data scaling software	SCALA
Phasing software	PHASER
Refinement software	PHENIX ((dev_2499: ???))

Data quality characteristics

	Overall
Low resolution limit [Å]	62.520
High resolution limit [Å]	1.800
Number of reflections	93996
<I/σ(I)>	5.41
Completeness [%]	83.6
Redundancy	1.7
CC(1/2)	0.960

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP		293	BAR protein was incubated with 1 mM acetyl-CoA for >2 hour prior to setting crystal trays. Crystals of BAR were obtained after 3 days at 20C in hanging drops containing 1 uL of protein solution (7.5 mg/mL) and 1 uL of reservoir solution (0.18 M calcium acetate, 0.1 M Tris-HCl pH 7, 18% (w/v) PEG 3000, 0.2% (v/v) N-nonyl Beta-D-glucopyranoside, 1 mM acetyl-CoA). Several crystals were soaked in reservoir solution supplemented with 30 mM L-phosphinothricin for 30-60 min before freezing. Crystals were frozen in reservoir solution supplemented with 15% (v/v) ethylene glycol. Acetylation of phosphinothricin occurred during soaking as no density for the acetyl group of acetyl-CoA was observed in the BAR/CoA/phosphinothricin ternary complex.