2OWO

Last Stop on the Road to Repair: Structure of E.coli DNA Ligase Bound to Nicked DNA-Adenylate

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 24-ID-C
Synchrotron site	APS
Beamline	24-ID-C
Temperature [K]	100
Detector technology	CCD
Collection date	2006-12-19
Detector	ADSC QUANTUM 4
Wavelength(s)	0.9792
Spacegroup name	C 1 2 1
Unit cell lengths	110.441, 99.273, 86.245
Unit cell angles	90.00, 105.33, 90.00

Refinement procedure

Resolution	30.340 - 2.300
Rwork	0.217
R-free	0.25400
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1. EfaLigA (aa 71-317 from PDB 1TA8) 2. TfiLigA (aa 433-584 and 321-391 from PDB 1V9P)
RMSD bond length	0.006
RMSD bond angle	1.200
Data reduction software	DENZO
Data scaling software	SCALEPACK
Phasing software	MOLREP
Refinement software	CNS (1.1)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	40.000	2.380
High resolution limit [Å]	2.300	2.300
Rmerge	0.084	0.422
Number of reflections	39100
<I/σ(I)>	12
Completeness [%]	98.7	91.2
Redundancy	4	3.5

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	6	295	EcoLigA (0.3 mM) was reacted with 5 mM MgCl2 and 0.3 mM NAD+ for 30 min at 22 C. The ligase-adenylylation reaction was then quenched by adding 10 mM EDTA. The mixture was supplemented with 26-bp nicked duplex DNA (0.318 mM). This LigA-nucleic acid solution was mixed 1:2 with a well solution containing 200 mM ammonium sulfate, 50 mM sodium acetate, 24% PEG-4000. Crystals were grown at 22 C by the sitting drop vapor diffusion method. Crystals appeared after 3 days. The crystals were transferred serially to solutions containing 5% glycerol/24% PEG-4000, 10% glycerol/26% PEG-4000, 15% glycerol/28% PEG-4000, and 20% glycerol/30% PEG-4000 in 100 mM ammonium sulfate, 50 mM sodium acetate, after which they were flash-frozen in liquid nitrogen. , pH 6.0, VAPOR DIFFUSION, SITTING DROP, temperature 295K

Crystallization Reagents

ID	crystal ID	solution ID	reagent name	concentration	details
1	1	1	sodium acetate
2	1	1	ammonium sulfate
3	1	1	PEG-400
4	1	2	sodium acetate
5	1	2	ammonium sulfate
6	1	2	PEG-400