6HUY
HmdII from Desulfurobacterium thermolithotrophum reconstitued with Fe-guanylylpyridinol (FeGP) cofactor and co-crystallized with methenyl-tetrahydrofolate form A
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | SYNCHROTRON |
Source details | SLS BEAMLINE X10SA |
Synchrotron site | SLS |
Beamline | X10SA |
Temperature [K] | 100 |
Detector technology | PIXEL |
Collection date | 2018-04-15 |
Detector | DECTRIS PILATUS 6M |
Wavelength(s) | 1.00000 |
Spacegroup name | P 21 21 21 |
Unit cell lengths | 99.769, 131.829, 168.263 |
Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
Resolution | 24.983 - 2.250 |
R-factor | 0.1881 |
Rwork | 0.187 |
R-free | 0.21220 |
Structure solution method | MOLECULAR REPLACEMENT |
Starting model (for MR) | 4yt4 |
RMSD bond length | 0.007 |
RMSD bond angle | 0.879 |
Data reduction software | XDS |
Data scaling software | SCALA (3.3.22) |
Phasing software | PHASER (2.6.0) |
Refinement software | PHENIX ((1.10.1_2155: ???)) |
Data quality characteristics
Overall | Outer shell | |
Low resolution limit [Å] | 48.890 | 2.370 |
High resolution limit [Å] | 2.250 | 2.250 |
Rmerge | 0.096 | 1.421 |
Rmeas | 0.108 | 1.595 |
Rpim | 0.049 | 0.714 |
Number of reflections | 105630 | 15270 |
<I/σ(I)> | 9.7 | |
Completeness [%] | 99.9 | 100 |
Redundancy | 4.8 | 4.9 |
CC(1/2) | 0.998 | 0.623 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, SITTING DROP | 5.5 | 281 | dHmdII reconstituted with the Fe-guanylylpyridinol cofactor from Methanothermobacter marburgensis was cocrystallized with methenyl-tetrahydrofolate using the sitting drop vapor diffusion method at 281 K under N2/H2 (95%/5%) in red light condition. The reconstituted dHmdII holoenzyme was mixed with methenyl-tetrahydrofolate 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 (with Fe-guanylylpryridinol and methenyl-tetrahydrofolate) 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 10 days, the crystals appeared in 20% PEG 3000 (w/v), 100 mM Tri-Sodium Citrate pH 5.5 (from the kit Wizard, Jean Bioscience). 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. |