3QI5
Crystal structure of human alkyladenine DNA glycosylase in complex with 3,N4-ethenocystosine containing duplex DNA
Experimental procedure
| Experimental method | SINGLE WAVELENGTH |
| Source type | SYNCHROTRON |
| Source details | ALS BEAMLINE 12.3.1 |
| Synchrotron site | ALS |
| Beamline | 12.3.1 |
| Temperature [K] | 100 |
| Detector technology | CCD |
| Collection date | 2006-07-11 |
| Detector | ADSC QUANTUM 315 |
| Wavelength(s) | 1.116 |
| Spacegroup name | P 1 |
| Unit cell lengths | 41.226, 41.220, 82.144 |
| Unit cell angles | 81.23, 88.40, 89.15 |
Refinement procedure
| Resolution | 81.000 - 2.200 |
| R-factor | 0.23881 |
| Rwork | 0.236 |
| R-free | 0.28427 |
| Structure solution method | MOLECULAR REPLACEMENT |
| Starting model (for MR) | 1bnk used as a search model |
| RMSD bond length | 0.005 |
| RMSD bond angle | 1.029 |
| 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] | 81.110 | 2.252 |
| High resolution limit [Å] | 2.200 | 2.200 |
| Number of reflections | 26278 | |
| Completeness [%] | 100.0 | 89.61 |
Crystallization Conditions
| crystal ID | method | pH | temperature | details |
| 1 | VAPOR DIFFUSION, HANGING DROP | 295 | The 3,N4-ethenocytosine (EDC) containing DNA duplex was prepared by annealing the EDC containing 13-mer crystallization oligonucleotide ('5-GAC ATG (EDC)TT GCC T-3') with its complementary strand that contained G opposite EDC (5'-GGC AAG CAT GTC A-3'). The delta79AAG-EDC complexes were prepared by mixing equimolar ratios of delta79AAG and EDC:G 13-mer DNA duplex at the final protein-DNA complex concentration of 0.3 mM in the complex buffer (20 mM Hepes-NaOH pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 5% v/v glycerol and 1 mM DTT). The complex was incubated on ice for 15 min and used for crystallization. The crystals were obtained upon mixing 1 uL of complex and 1 ul of the reservoir solution (100 mM sodium cacodylate pH 6.0, 200 mM manganese chloride and 20% polyethylene glycol (PEG)-3350) over 0.5 ml of the reservoir solution, followed by incubation for 2 days, VAPOR DIFFUSION, HANGING DROP, temperature 295K |
