1WCI

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-BM
Synchrotron site	APS
Beamline	19-BM
Temperature [K]	100
Detector technology	CCD
Collection date	2003-06-21
Detector	CUSTOM
Spacegroup name	P 31 2 1
Unit cell lengths	145.044, 145.044, 69.510
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	30.000 - 1.840
R-factor	0.149
Rwork	0.148
R-free	0.18800
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1ols
RMSD bond length	0.020
RMSD bond angle	1.662
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]	22.830	1.870
High resolution limit [Å]	1.840	1.840
Rmerge	0.080	0.800
Number of reflections	71058
<I/σ(I)>	22.5	1.9
Completeness [%]	97.3	80.8
Redundancy	6	5.2

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION	5.5	293	CRYSTALS WERE GROWN AT 20C VIA THE VAPOR DIFFUSION METHOD BY MIXING EQUAL AMOUNTS OF PROTEIN (20-25 MG/ML IN 50 MM HEPES/NAOH, PH 7.5, 250 MM KCL, 0.5 MM PMSF, 1 MM BENZAMIDINE AND 5% (V/V) GLYCEROL) WITH WELL SOLUTION (1.4-1.6 M AMMONIUM SULFATE, 0.1 M NA-CITRATE PH 5.8, 20 MM B-MERCAPTOETHANOL). SERIALLY DILUTED CRUSHED CRYSTALS WERE USED FOR MICRO-SEEDING ONE DAY AFTER THE DROPS WERE SET UP. CRYSTALS APPEARED ONE DAY AFTER SEEDING AND GREW TO A MAXIMUM SIZE OF 120 X 800 UM WITHIN 10 DAYS. CRYSTALS WERE STABILIZED FOR 12 HOURS BY TRANSFER TO FRESH WELL SOLUTION. THEY WERE THEN CRYO-PROTECTED BY STEP-WISE TRANSFER INTO CRYO-BUFFER CONTAINING 1.6 M AMMONIUM SULFATE, 50 MM HEPES, PH 7.5, 100 MM NA-CITRATE, PH 5.8, 100 MM KCL, 50 MM DTT AND UP TO 20% (V/V) GLYCEROL. IT WAS FOUND THAT MN2 IONS COULD REPLACE THE MG2 REQUIRED FOR THE BINDING OF THDP TO THE ENZYME.