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 |
