1SKU

E. coli Aspartate Transcarbamylase 240's Loop Mutant (K244N)

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	NSLS BEAMLINE X12C
Synchrotron site	NSLS
Beamline	X12C
Temperature [K]	100
Detector technology	CCD
Collection date	2003-12-30
Detector	BRANDEIS - B4
Wavelength(s)	1.00704
Spacegroup name	H 3
Unit cell lengths	125.670, 125.670, 198.200
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	29.550 - 2.600
R-factor	0.20186
Rwork	0.201
R-free	0.23117
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	A consensus model using overlayed previously published pdb entry 1EZZ (mutant P268A catalytic chain) and 1NBE (T82A regulatory chain) and wild type 3AT1 was used.
RMSD bond length	0.063
RMSD bond angle	4.586
Data reduction software	DENZO
Data scaling software	SCALEPACK
Phasing software	AMoRE
Refinement software	REFMAC (5.1.24)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	30.000	2.680
High resolution limit [Å]	2.590	2.590
Rmerge	0.039	0.327
Number of reflections	34988
<I/σ(I)>	13.3	12.2
Completeness [%]		89.8
Redundancy	6.5	2.62

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	7	298	Prior to crystallization the enzyme was dialyzed into 40 mM KH2PO4, 2.0 mM 2-mercaptoethanol, 0.2 mM EDTA, pH 7.0 for 24 hours. Single crystals of the K244N mutant were obtained by mixing a 20 mg/ml filtered solution (0.22 m) of the K244N enzyme with a solution of 17% (w/v) PEG 1450, 50 mM malonate, 0.2 mM EDTA, 1 mM sodium azide and 20 mM Bis-Tris buffer pH 7.0 in a 1:1 ratio (v/v), VAPOR DIFFUSION, HANGING DROP, temperature 298K