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 |
