1OLU

Roles of His291-alpha and His146-beta' in the reductive acylation reaction catalyzed by human branched-chain alpha-ketoacid dehydrogenase

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	2003-03-15
Spacegroup name	P 31 2 1
Unit cell lengths	145.379, 145.379, 69.497
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	23.790 - 1.900
R-factor	0.161
Rwork	0.161
R-free	0.20000
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1ols
RMSD bond length	0.014
RMSD bond angle	1.600
Data reduction software	HKL-2000
Data scaling software	HKL-2000
Phasing software	CNS (1.1)
Refinement software	CNS (1.1)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	23.790	1.930
High resolution limit [Å]	1.900	1.900
Rmerge	0.106	0.544
Number of reflections	61707
<I/σ(I)>	17.7	3.7
Completeness [%]	93.1	95.4
Redundancy	6.8	6.6

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.