6HAE
Crystal structure of [Fe]-hydrogenase (Hmd) from Methanococcus aeolicus in complex with FeGP cofactor and methenyl-tetrahydromethanopterin (close form B)
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 | 2017-07-06 |
Detector | DECTRIS PILATUS 6M |
Wavelength(s) | 0.99992 |
Spacegroup name | P 21 21 2 |
Unit cell lengths | 80.015, 156.480, 53.654 |
Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
Resolution | 44.560 - 1.850 |
R-factor | 0.163 |
Rwork | 0.161 |
R-free | 0.19200 |
Structure solution method | MOLECULAR REPLACEMENT |
Starting model (for MR) | 6hac |
RMSD bond length | 0.010 |
RMSD bond angle | 1.110 |
Data reduction software | XDS |
Data scaling software | SCALA (3.3.22) |
Phasing software | PHASER |
Refinement software | BUSTER (2.10.3) |
Data quality characteristics
Overall | Outer shell | |
Low resolution limit [Å] | 44.560 | 1.950 |
High resolution limit [Å] | 1.850 | 1.850 |
Rmerge | 0.124 | 1.133 |
Rmeas | 0.135 | 1.301 |
Rpim | 0.055 | 0.627 |
Number of reflections | 56771 | 6994 |
<I/σ(I)> | 8.6 | 1.2 |
Completeness [%] | 97.4 | 85 |
Redundancy | 6 | 4.1 |
CC(1/2) | 0.997 | 0.323 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, SITTING DROP | 7 | 298 | Crystallization of [Fe]-hydrogenase-methenyl-H4MPT+ complex was performed in an anaerobic tent with gas phase 100%N2 at room temperature under dark condition. The reconstituted [Fe]-hydrogenase holoenzyme (50 mg/ml) was mixed with 10-mM methenyl-H4MPT+, both of which contained 10-mM MOPS/KOH pH 7.0. The final concentrations of [Fe]-hydrogenase and methenyl-H4MPT+ were 24 mg/ml and 3 mM, respectively. After incubating the mixture in this tent under dark condition for 5 min, the enzyme solution was centrifuged using MiniSpin-plus (Eppendorf) at 8000 rpm for 5 min by using centrifugal filters made of polyvinylidene fluoride (PVDF, Millipore). The crystallization solution contained 20 % w/v polyethylene glycol 3350, 200 mM sodium thiocyanate and 3% w/v 1,5-diaminopentane dihydrochloride with a ratio of protein mixture and crystallization reservoir of 0.7 ul / 0.7 ul drops (in 96 well-plates). |