6HUZ
HmdII from Desulfurobacterium thermolithotrophum reconstituted with Fe-guanylylpyridinol (FeGP) cofactor and co-crystallized with methenyl-tetrahydrofolate form B
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | SYNCHROTRON |
Source details | SOLEIL BEAMLINE PROXIMA 1 |
Synchrotron site | SOLEIL |
Beamline | PROXIMA 1 |
Temperature [K] | 100 |
Detector technology | PIXEL |
Collection date | 2018-06-07 |
Detector | DECTRIS PILATUS 6M |
Wavelength(s) | 0.97857 |
Spacegroup name | P 21 21 2 |
Unit cell lengths | 65.200, 132.020, 49.160 |
Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
Resolution | 46.390 - 1.850 |
R-factor | 0.196 |
Rwork | 0.195 |
R-free | 0.22300 |
Structure solution method | MOLECULAR REPLACEMENT |
Starting model (for MR) | 6huy |
RMSD bond length | 0.010 |
RMSD bond angle | 1.140 |
Data reduction software | XDS |
Data scaling software | SCALA (3.3.22) |
Phasing software | PHASER (2.6.0) |
Refinement software | BUSTER (2.10.3) |
Data quality characteristics
Overall | Outer shell | |
Low resolution limit [Å] | 49.160 | 1.950 |
High resolution limit [Å] | 1.850 | 1.850 |
Rmerge | 0.085 | 1.212 |
Rmeas | 0.092 | 1.317 |
Rpim | 0.036 | 0.511 |
Number of reflections | 37060 | 5308 |
<I/σ(I)> | 10.2 | 1.2 |
Completeness [%] | 99.9 | 99.7 |
Redundancy | 6.5 | 6.6 |
CC(1/2) | 0.998 | 0.849 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, SITTING DROP | 5.5 | 281 | HmdII from Desulfurobacterium thermolithotrophum was reconstituted with the Fe-guanylylpyridinol cofactor from Methanothermobacter marburgensis and co-crystallized with methenyl-tetrahydrofolate using the sitting drop vapor diffusion method under N2/H2 (95%/5%) in red light condition. The reconstituted holoenzyme was mixed with methenyl-H4F+ at the final concentrations of 1.6 mM. Methenyl-tetrahydrofolate was dissolved in 10% DMSO, and the final concentration of DMSO in the protein solution was 1.6%. 0.7 ul of dHmdII at 21 mg/ml (reconstituted with FeGP and methenyl-H4F+) was spotted on a 96-well 2-drop MRC Crystallization Plates (Molecular Dimensions, Suffolk, UK) and 0.7 ul of reservoir solution was mixed. After one month, crystals appeared in 20% PEG 3000 (w/v) and 100 mM Sodium Citrate pH 5.5 (from the kit WIZARD, Jena Bioscience). The crystals were cryoprotected by a soak of few seconds in 20% PEG 3000 (w/v), 100 mM Tri-Sodium Citrate pH 5.5 and 20% glycerol before a flash freeze in liquid nitrogen |