1OLS

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	ROTATING ANODE
Source details	RIGAKU RU300
Temperature [K]	100
Detector technology	IMAGE PLATE
Collection date	2002-02-07
Detector	RIGAKU-MSC
Spacegroup name	P 31 2 1
Unit cell lengths	144.721, 144.721, 69.151
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	39.090 * - 1.850
R-factor	0.172
Rwork	0.172
R-free	0.20180 *
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1dtw
RMSD bond length	0.008 *
RMSD bond angle	1.360 *
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]	39.090 *	1.880
High resolution limit [Å]	1.850	1.850
Rmerge	0.048	0.556
Total number of observations	313991 *
Number of reflections	71059
<I/σ(I)>	27.8	2.5
Completeness [%]	99.8	99.7
Redundancy	4.5	4

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION	7.5 *	20 *	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.

Crystallization Reagents in Literatures

ID	crystal ID	solution	reagent name	concentration (unit)	details
1	1	drop	protein	20-25 (mg/ml)
10	1	reservoir	beta-mercaptoethanol	20 (mM)
2	1	drop	sodium HEPES	50 (mM)	pH7.5
3	1	drop		250 (mM)
4	1	drop	phenylmethylsulfonyl fluoride	0.5 (mM)
5	1	drop	benzamidine	1 (mM)
6	1	drop	dithiothreitol	20 (mM)
7	1	drop	glycerol	5 (%(v/v))
8	1	reservoir	ammonium sulfate	1.4-1.6 (M)
9	1	reservoir	sodium citrate	0.1 (M)	pH5.8