2R0C

Structure of the substrate-free form of the rebeccamycin biosynthetic enzyme REBC

Experimental procedure

Experimental method	MAD
Source type	SYNCHROTRON
Source details	SSRL BEAMLINE BL9-2
Synchrotron site	SSRL
Beamline	BL9-2
Temperature [K]	100
Detector technology	CCD
Collection date	2006-07-09
Detector	MARMOSAIC 325 mm CCD
Wavelength(s)	1.8455, 1.8447, 0.9798
Spacegroup name	P 1 21 1
Unit cell lengths	63.220, 77.562, 64.673
Unit cell angles	90.00, 108.04, 90.00

Refinement procedure

Resolution	50.000 - 1.800
R-factor	0.211
Rwork	0.211
R-free	0.24100
Structure solution method	MAD
RMSD bond length	0.006
RMSD bond angle	1.280
Data reduction software	HKL-2000
Data scaling software	SCALEPACK
Phasing software	SHARP
Refinement software	CNS (1.1)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	50.000	1.860
High resolution limit [Å]	1.800	1.800
Number of reflections	52005
<I/σ(I)>	25.4	2.6
Completeness [%]	93.9	67.2
Redundancy	7.2

Crystallization Conditions

crystal ID	method	pH	temperature	details
1		7.4	298	1.5 microliters of RebC (9 mg/mL in 150 mM NaCl, 10% glycerol, 25 mM HEPES pH 7.5) were incubated with 0.35 microliters of guanidine-HCl for 30 seconds, followed by addition of 1.5 microliters of precipitant solution (19% PEG-8000, 0.1 M HEPES pH 7.4), without mixing, at room temperature and sealed over a precipitant well solution. Immediately after set up, crystal trays were placed on a gel shaker and then, after 12 hours, transferred to a storage space in vibration-isolation. Before being flash-frozen in liquid nitrogen, a crystal was soaked for five seconds in cryogenic solution (19% PEG-8000, 0.1 M HEPES pH 7.4, 20% glycerol), VAPOR DIFFUSION, HANGING DROP, temperature 298K, pH 7.40