2BFD

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

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 19-ID
Synchrotron site	APS
Beamline	19-ID
Temperature [K]	100
Detector technology	CCD
Collection date	2004-04-12
Detector	CUSTOM
Spacegroup name	P 31 2 1
Unit cell lengths	144.839, 144.839, 69.247
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	30.000 - 1.390
R-factor	0.15
Rwork	0.149
R-free	0.17000
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1ols
RMSD bond length	0.021
RMSD bond angle	2.086
Data reduction software	HKL-2000
Data scaling software	HKL-2000
Refinement software	REFMAC (5.2.0003)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	20.370	1.420
High resolution limit [Å]	1.400	1.400
Rmerge	0.070	0.670
Number of reflections	161893
<I/σ(I)>	19.8	1.9
Completeness [%]	97.2	97.2
Redundancy	9.4	4.4

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION	5.5	295	CRYSTALS 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).