2YCT

Tyrosine phenol-lyase from Citrobacter freundii in complex with pyridine N-oxide and the quinonoid intermediate formed with L-alanine

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	ROTATING ANODE
Source details	RIGAKU RU200
Temperature [K]	100
Detector technology	IMAGE PLATE
Collection date	1999-09-21
Detector	MARRESEARCH
Spacegroup name	P 21 21 2
Unit cell lengths	133.993, 143.770, 60.081
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	20.000 - 2.250
R-factor	0.14003
Rwork	0.139
R-free	0.18110
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	2ez2
RMSD bond length	0.014
RMSD bond angle	1.335
Data reduction software	DENZO
Data scaling software	SCALEPACK
Phasing software	AMoRE
Refinement software	REFMAC (5.4.0077)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	20.000	2.290
High resolution limit [Å]	2.250	2.250
Rmerge	0.050	0.170
Number of reflections	55230
<I/σ(I)>	13.8	4.5
Completeness [%]	99.0	94
Redundancy	3.4	2.8

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 ALANINE QUINONOID COMPLEX WITH PYRIDINE N-OXIDE (9PO) WAS PREPARED BY SOAKING WILD TYPE TPL CRYSTALS IN THE STABILIZATION SOLUTION CONTAINING 40% (W/V) POLY(ETHYLENE GLYCOL) 5000 MONOMETHYL ETHER (PEG 5000 MME), 50 MM TRIETHANOLAMINE PH 8.0, 0.25 M KCL, 0.2 MM PYRIDOXAL 5-PHOSPHATE (PLP), 0.5 MM DITHIOTHREITOL (DTT), WITH ADDITION OF 100 MM L- ALANINE AND A SATURATING CONCENTRATION OF PYRIDINE N-OXIDE. SINCE THE CRYSTALS QUALITY DETERIORATED RAPIDLY, THE SOAKING TIME WAS RESTRICTED TO ABOUT 20 S.