2VLH

Quinonoid intermediate of Citrobacter freundii tyrosine phenol-lyase formed with methionine

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ESRF BEAMLINE BM14
Synchrotron site	ESRF
Beamline	BM14
Temperature [K]	120
Detector technology	CCD
Collection date	2005-04-29
Detector	MARRESEARCH
Spacegroup name	P 21 21 2
Unit cell lengths	132.886, 143.284, 59.665
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	97.590 - 1.950
R-factor	0.156
Rwork	0.155
R-free	0.19200
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	2ez2
RMSD bond length	0.015
RMSD bond angle	1.399
Data reduction software	DENZO
Data scaling software	SCALEPACK
Phasing software	AMoRE
Refinement software	REFMAC (5.2.0019)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	30.000	2.020
High resolution limit [Å]	1.950	1.950
Rmerge	0.080	0.420
Number of reflections	76260
<I/σ(I)>	10.5
Completeness [%]	90.3	82.3
Redundancy	3.9	3.6

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	8	293	CRYSTALS OF THE C. FREUNDII TPL WERE GROWN AT 277 AND 293 K USING THE HANGING DROP VAPOR DIFFUSION METHOD. THE BEST CRYSTALS WERE OBTAINED BY MIXING 2 UL OF THE PROTEIN SOLUTION (18-20 MG/ML) CONTAINING 50MM K-PHOSPHATE PH 8.0, 0.5 MM PLP, 1MM DDT WITH AN EQUAL VOLUME OF THE RESERVOIR SOLUTION CONTAINING 50 MM TRIETHANOLAMINE BUFFER (PH 8.0), 0.5 MM PLP, 2 MM DDT, 0.4 M KCL, AND 35-38% (W/V) POLY(ETHYLENE GLYCOL) 5000 MONOMETHYL ETHER. THE INTERMEDIATE WITH METHIONINE WAS PREPARED BY SOAKING THE TPL CRYSTALS FOR 5 MIN IN THE STABILIZATION SOLUTION CONTAINING 40% POLY(ETHYLENE GLYCOL) 5000 MONOMETHYL ETHER, 50 MM TRIETHANOLAMINE BUFFER (PH 8.0), 0.25 M KCL, 0.2 MM PLP, 0.5 MM DTT AND 70 MM L-METHIONINE. CRYSTALS WERE FROZEN DIRECTLY FROM THE SOAKING SOLUTION.