3EPT

Structure of the rebeccamycin biosynthetic enzyme RebC with reduced flavin

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	SSRL BEAMLINE BL9-2
Synchrotron site	SSRL
Beamline	BL9-2
Temperature [K]	100
Detector technology	CCD
Collection date	2007-07-22
Detector	MARMOSAIC 325 mm CCD
Spacegroup name	P 1 21 1
Unit cell lengths	63.261, 78.223, 123.063
Unit cell angles	90.00, 98.66, 90.00

Refinement procedure

Resolution	40.000 - 2.970
R-factor	0.225
Rwork	0.225
R-free	0.27600
Structure solution method	RIGID BODY
Starting model (for MR)	2r0g
RMSD bond length	0.007
RMSD bond angle	1.340
Data reduction software	HKL-2000
Data scaling software	SCALEPACK
Phasing software	CNS
Refinement software	CNS

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	40.000	3.080
High resolution limit [Å]	2.970	2.970
Number of reflections	22485
<I/σ(I)>		3
Completeness [%]	90.4	50.7
Redundancy	4.1	3.1

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. Reduced-RebC was generated by incubating a RebC crystal in a cryogenic solution (19% PEG-8000, 0.1 M HEPES pH 7.4, 20% glycerol) and adding an amount of solid sodium dithionite that is in slight excess of the point at which the crystal became completely clear, indicating that all FAD was reduced. The crystal was then flash-frozen in liquid nitrogen, VAPOR DIFFUSION, HANGING DROP, temperature 298K