6HAE

Crystal structure of [Fe]-hydrogenase (Hmd) from Methanococcus aeolicus in complex with FeGP cofactor and methenyl-tetrahydromethanopterin (close form B)


X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
TypeSourceAccession CodeDetails
experimental modelPDB 6HAC 

Crystallization

Crystalization Experiments
IDMethodpHTemperatureDetails
1VAPOR DIFFUSION, SITTING DROP7298Crystallization 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).
Crystal Properties
Matthews coefficientSolvent content
2.2946.22

Crystal Data

Unit Cell
Length ( Å )Angle ( ˚ )
a = 80.015α = 90
b = 156.48β = 90
c = 53.654γ = 90
Symmetry
Space GroupP 21 21 2

Diffraction

Diffraction Experiment
ID #Crystal IDScattering TypeData Collection TemperatureDetectorDetector TypeDetailsCollection DateMonochromatorProtocol
11x-ray100PIXELDECTRIS PILATUS 6M2017-07-06MSINGLE WAVELENGTH
Radiation Source
ID #SourceTypeWavelength ListSynchrotron SiteBeamline
1SYNCHROTRONSLS BEAMLINE X10SA0.99992SLSX10SA

Data Collection

Overall
ID #Resolution (High)Resolution (Low)Percent Possible (Observed)R Merge I (Observed)Rrim I (All)Rpim I (All)CC (Half)Net I Over Average Sigma (I)RedundancyNumber Reflections (All)Number Reflections (Observed)Observed Criterion Sigma (F)Observed Criterion Sigma (I)B (Isotropic) From Wilson Plot
11.8544.5697.40.1240.1350.0550.9978.665677126.94
Highest Resolution Shell
ID #Resolution (High)Resolution (Low)Percent Possible (All)Percent Possible (Observed)R Merge I (Observed)Rrim I (All)Rpim I (All)CC (Half)Mean I Over Sigma (Observed)RedundancyNumber Unique Reflections (All)
11.851.95851.1331.3010.6270.3231.24.1

Refinement

Statistics
Diffraction IDStructure Solution MethodCross Validation methodStarting modelResolution (High)Resolution (Low)Number Reflections (Observed)Number Reflections (R-Free)Percent Reflections (Observed)R-Factor (Observed)R-WorkR-FreeR-Free Selection DetailsMean Isotropic B
X-RAY DIFFRACTIONMOLECULAR REPLACEMENTTHROUGHOUT6HAC1.8544.5656710280497.10.1630.1610.192RANDOM28.83
Temperature Factor Modeling
Anisotropic B[1][1]Anisotropic B[1][2]Anisotropic B[1][3]Anisotropic B[2][2]Anisotropic B[2][3]Anisotropic B[3][3]
3.8163-1.9962-1.8201
RMS Deviations
KeyRefinement Restraint Deviation
t_other_torsion12.5
t_omega_torsion2.83
t_angle_deg1.11
t_bond_d0.01
t_dihedral_angle_d
t_incorr_chiral_ct
t_pseud_angle
t_trig_c_planes
t_gen_planes
t_it
RMS Deviations
KeyRefinement Restraint Deviation
t_other_torsion12.5
t_omega_torsion2.83
t_angle_deg1.11
t_bond_d0.01
t_dihedral_angle_d
t_incorr_chiral_ct
t_pseud_angle
t_trig_c_planes
t_gen_planes
t_it
t_nbd
t_improper_torsion
t_chiral_improper_torsion
t_sum_occupancies
t_utility_distance
t_utility_angle
t_utility_torsion
t_ideal_dist_contact
Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen AtomsNumber
Protein Atoms5132
Nucleic Acid Atoms
Solvent Atoms592
Heterogen Atoms264

Software

Software
Software NamePurpose
BUSTERrefinement
XDSdata reduction
SCALAdata scaling
PHASERphasing