2W3V

MYCOBACTERIUM AVIUM DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND TRIMETHOPRIM


X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
IDMethodpHTemperatureDetails
1VAPOR DIFFUSION, HANGING DROP6.5277MAVDHFR (15 MG/ML) IN 20 MM HEPES (PH 7.0), 1 MM DTT, 0.1 MM EDTA, 1.5 MM NAN3 WAS MIXED WITH NADPH (3 MM, 15 MIN AT ROOM TEMPERATURE) AND THEN TRIMETHOPRIM (2 MM, 15 MIN ON ICE). CRYSTALLIZATION (HANGING DROP VAPOR PHASE EQUILIBRATION) WAS ACHIEVED BY MIXING WITH AN EQUAL VOLUME OF THE PROTEIN COMPLEX WITH A RESERVOIR SOLUTION CONSISTING OF 70% 2-METHYL-2, 4-PENTANEDIOL (MPD) AND 100 MM HEPES (PH 6.5), AND SUSPENDING THE MIXTURE OVER THE RESERVOIR AT 277 K. SMALL ROD-LIKE CRYSTALS (0.01 X 0.01 X 0.05 MM) GREW WITHIN 2 DAYS. CRYSTALS WERE FLASH-COOLED DIRECTLY FROM THE DROP IN LIQUID N2.
Crystal Properties
Matthews coefficientSolvent content
2.550.9

Crystal Data

Unit Cell
Length ( Å )Angle ( ˚ )
a = 70.816α = 90
b = 70.816β = 90
c = 73.613γ = 90
Symmetry
Space GroupP 41 21 2

Diffraction

Diffraction Experiment
ID #Crystal IDScattering TypeData Collection TemperatureDetectorDetector TypeDetailsCollection DateMonochromatorProtocol
11x-ray100IMAGE PLATEMARRESEARCHMIRRORS2000-01-20MSINGLE WAVELENGTH
Radiation Source
ID #SourceTypeWavelength ListSynchrotron SiteBeamline
1SYNCHROTRONSSRL BEAMLINE BL9-10.785SSRLBL9-1

Data Collection

Overall
ID #Resolution (High)Resolution (Low)Percent Possible (Observed)R Merge I (Observed)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
11.891598.20.0711.24.615319-1024.8
Highest Resolution Shell
ID #Resolution (High)Resolution (Low)Percent Possible (All)Percent Possible (Observed)R Merge I (Observed)Mean I Over Sigma (Observed)RedundancyNumber Unique Reflections (All)
11.891.9590.50.542.74.1

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 REPLACEMENTTHROUGHOUTPDB ENTRY 1RX11.8914.831452977398.30.1820.180.224RANDOM29.52
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]
1.171.17-2.33
RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg29.172
r_dihedral_angle_4_deg16.73
r_dihedral_angle_3_deg12.153
r_dihedral_angle_1_deg5.902
r_scangle_it3.31
r_scbond_it2.274
r_angle_refined_deg1.553
r_mcangle_it1.395
r_mcbond_it0.904
r_nbtor_refined0.31
RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg29.172
r_dihedral_angle_4_deg16.73
r_dihedral_angle_3_deg12.153
r_dihedral_angle_1_deg5.902
r_scangle_it3.31
r_scbond_it2.274
r_angle_refined_deg1.553
r_mcangle_it1.395
r_mcbond_it0.904
r_nbtor_refined0.31
r_symmetry_vdw_refined0.2
r_nbd_refined0.197
r_xyhbond_nbd_refined0.153
r_symmetry_hbond_refined0.146
r_chiral_restr0.104
r_bond_refined_d0.014
r_gen_planes_refined0.006
r_bond_other_d
r_angle_other_deg
r_gen_planes_other
r_nbd_other
r_nbtor_other
r_xyhbond_nbd_other
r_metal_ion_refined
r_metal_ion_other
r_symmetry_vdw_other
r_symmetry_hbond_other
r_symmetry_metal_ion_refined
r_symmetry_metal_ion_other
r_mcbond_other
r_mcangle_other
r_scbond_other
r_scangle_other
r_long_range_B_refined
r_long_range_B_other
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded
Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen AtomsNumber
Protein Atoms1276
Nucleic Acid Atoms
Solvent Atoms160
Heterogen Atoms69

Software

Software
Software NamePurpose
REFMACrefinement
MOSFLMdata reduction
SCALAdata scaling
AMoREphasing