1V1M

CROSSTALK BETWEEN COFACTOR BINDING AND THE PHOSPHORYLATION LOOP CONFORMATION IN THE BCKD MACHINE

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	2003-07-25
Detector	ADSC CCD
Spacegroup name	P 31 2 1
Unit cell lengths	145.287, 145.287, 69.165
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	28.870 - 2.000
R-factor	0.13
Rwork	0.129
R-free	0.17000
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1ols
RMSD bond length	0.020
RMSD bond angle	1.700
Data reduction software	HKL-2000
Data scaling software	HKL-2000
Phasing software	REFMAC (5.1)
Refinement software	REFMAC (5.1.24)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	36.320	2.030
High resolution limit [Å]	2.000	2.000
Rmerge	0.117	0.680
Number of reflections	54331
<I/σ(I)>	16.5	3.2
Completeness [%]	95.4	96.8
Redundancy	6.2	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.