2YCP

F448H mutant of tyrosine phenol-lyase from Citrobacter freundii in complex with quinonoid intermediate formed with 3-fluoro-L-tyrosine

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ESRF BEAMLINE BM30A
Synchrotron site	ESRF
Beamline	BM30A
Temperature [K]	100
Detector technology	CCD
Collection date	2005-05-01
Detector	MARRESEARCH SX-165
Spacegroup name	P 21 21 21
Unit cell lengths	136.423, 143.757, 118.543
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	30.000 - 2.000
R-factor	0.14114
Rwork	0.141
R-free	0.17470
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	2ez2
RMSD bond length	0.013
RMSD bond angle	1.304
Data reduction software	DENZO
Data scaling software	SCALEPACK
Phasing software	PHASER
Refinement software	REFMAC (5.5.0109)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	30.000	2.070
High resolution limit [Å]	2.000	2.000
Rmerge	0.090	0.530
Number of reflections	154478
<I/σ(I)>	7.5	2.5
Completeness [%]	99.0	99
Redundancy	4.7	4.5

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	8	293	CRYSTALS OF THE C. FREUNDII F448H 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 50 MM K-PHOSPHATE PH 8.0, 0.5 MM PLP, 1 MM 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 QUINONOID INTERMEDIATE WITH 3-FLUORO-L-TYROSINE WAS PREPARED BY SOAKING THE TPL CRYSTALS FOR ABOUT 30 SECONDS 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 10 MM 3-FLUORO-L-TYROSINE. CRYSTALS WERE FROZEN DIRECTLY FROM THE SOAKING SOLUTION.