2BFE | pdb_00002bfe

Reactivity modulation of human branched-chain alpha-ketoacid dehydrogenase by an internal molecular switch


X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
TypeSourceAccession CodeDetails
experimental modelPDB 1OLSPDB ENTRY 1OLS

Crystallization

Crystalization Experiments
IDMethodpHTemperatureDetails
1VAPOR DIFFUSION5.5295CRYSTALS WERE GROWN AT 22C VIA THE VAPOR DIFFUSION METHOD IN COMPLEX WITH A 40 AMINO ACID PEPTIDE DERIVED FROM THE SUBUNIT BINDING DOMAIN (SBD) OF THE E2 COMPONENT OF BRANCHED CHAIN ALPHA-KETOACID DEHDROGENASE. THIS COMPLEX WAS FORMED BY MIXING N-TERMINALLY HIS6-TAGGED PROTEIN WITH C-TERMINALLY HIS6-TAGGED SBD IN 50 MM NA-HEPES, PH 7.5, 150 MM KCL, 20 MM DTT AND 5% (V/V) GLYCEROL AT A MOLAR RATIO OF 1:4. CRYSTALS OF THE COMPLEX (20 MG/ML) WERE OBTAINED BY MIXING 3 MICROLITERS OF PROTEIN WITH 3 MICROLITERS OF CRYSTALLIZATION SOLUTION (10% (V/V) POLYETHYLENE GLYCOL 4000, 10% (V/V) MPD AND 0.1M SODIUM CITRATE, PH 5.8) WITH 1 ML OF CRYSTALLIZATION SOLUTION IN THE RESERVOIR. MANGANESE IONS WERE USED INSTEAD OF MAGNESIUM REQUIRED FOR THE BINDING OF THIAMIN DIPHOSPHATE TO THE ENZYME. THE PRESENCE OF MANGANESE IONS IN THE CRYSTALS RESULTED IN IMPROVED X-RAY DIFFRACTION QUALITIES WITHOUT AFFECTING THE CATALYTIC PROPERTIES. CRYSTALS WERE CRYO-PROTECTED BY STEP-WISE TRANSFER INTO CRYO-BUFFER (CRYSTALLIZATION SOLUTION CONTAINING 5-10%(V/V) GLYCEROL).
Crystal Properties
Matthews coefficientSolvent content
2.553.4

Crystal Data

Unit Cell
Length ( Å )Angle ( ˚ )
a = 145.181α = 90
b = 145.181β = 90
c = 69.573γ = 120
Symmetry
Space GroupP 31 2 1

Diffraction

Diffraction Experiment
ID #Crystal IDScattering TypeData Collection TemperatureDetectorDetector TypeDetailsCollection DateMonochromatorProtocol
11x-ray100CCDADSC CCD2004-06-04MSINGLE WAVELENGTH
Radiation Source
ID #SourceTypeWavelength ListSynchrotron SiteBeamline
1SYNCHROTRONAPS BEAMLINE 19-IDAPS19-ID

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
11.6939.2499.10.0628.65.893358-319.02
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)
11.691.7288.10.441.93.2

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-Work (Depositor)R-Work (DCC)R-Free (Depositor)R-Free (DCC)R-Free Selection DetailsMean Isotropic B
X-RAY DIFFRACTIONMOLECULAR REPLACEMENTTHROUGHOUTPDB ENTRY 1OLS1.693091818150499.10.150.150.170.1810.19RANDOM18.66
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]
0.990.490.99-1.48
RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg35.336
r_dihedral_angle_4_deg18.229
r_dihedral_angle_3_deg14.313
r_dihedral_angle_1_deg6.11
r_scangle_it4.653
r_scbond_it2.978
r_angle_refined_deg1.754
r_mcangle_it1.678
r_mcbond_it0.985
r_nbtor_refined0.314
Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen AtomsNumber
Protein Atoms5551
Nucleic Acid Atoms
Solvent Atoms612
Heterogen Atoms44

Software

Software
Software NamePurpose
REFMACrefinement
HKL-2000data reduction
HKL-2000data scaling