3GTS

Crystal Structure of Dicamba Monooxygenase with Non-heme Iron and Dicamba

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 22-ID
Synchrotron site	APS
Beamline	22-ID
Temperature [K]	100
Detector technology	CCD
Collection date	2005-06-30
Detector	MARMOSAIC 300 mm CCD
Wavelength(s)	1.000
Spacegroup name	P 32
Unit cell lengths	80.800, 80.800, 159.600
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	20.000 - 2.200
Rwork	0.230
R-free	0.26600
Starting model (for MR)	MR was performed using a preliminary DMO structure. This early DMO structure was obtained largely using a high-redundancy 1.5418 A wavelength data set for Fe single wavelength anomalous dispersion (SAD) phasing with assistance from the sulfur anomalous signal in a high-redundancy 2.29 A wavelength data set.
RMSD bond length	0.006
RMSD bond angle	1.320
Data reduction software	HKL-2000
Data scaling software	HKL-2000
Phasing software	PHASER
Refinement software	CNX

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	36.040	2.280
High resolution limit [Å]	2.200	2.200
Rmerge	0.056	0.419
Number of reflections	58874
<I/σ(I)>	27.6
Completeness [%]	99.7	100
Redundancy	4	4

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	5.5	294	The purified protein was concentrated to 10-20 mg/ml and buffer exchanged into 25 mM Tris-HCl, pH 8.0, 30-50 mM NaCl, 25 mM sodium citrate. DMO crystals were grown using the vapor diffusion by sitting drop method, with a reservoir solution of 8-11% PEG 6000, 100 mM sodium acetate buffer-pH 5.5, 1 M LiCl, and 4ul droplets of varying protein-to-precipitant ratios. To prepare a DMO-non-heme iron-dicamba crystal, some crystals were transferred to a cryo-amenable storage solution that contained 25 mM dicamba (12.3 % PEG6000, 95 mM sodium acetate pH 5.5, 21% glycerol, 1mM NaN3, and 25 mM dicamba) for 5-6 hours prior to plunge-cooling for cryo-storage and low temperature data collection. VAPOR DIFFUSION, SITTING DROP, temperature 294K