2AJQ
Structure of replicative DNA polymerase provides insigts into the mechanisms for processivity, frameshifting and editing
Experimental procedure
| Experimental method | SINGLE WAVELENGTH |
| Source type | SYNCHROTRON |
| Source details | NSLS BEAMLINE X26C |
| Synchrotron site | NSLS |
| Beamline | X26C |
| Temperature [K] | 100 |
| Detector technology | CCD |
| Collection date | 2005-03-28 |
| Detector | ADSC QUANTUM 4 |
| Wavelength(s) | 1.100 |
| Spacegroup name | C 2 2 21 |
| Unit cell lengths | 168.312, 169.235, 179.788 |
| Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
| Resolution | 45.970 - 2.600 |
| R-factor | 0.2331 |
| Rwork | 0.233 |
| R-free | 0.28400 |
| Structure solution method | MOLECULAR REPLACEMENT |
| RMSD bond length | 0.007 |
| RMSD bond angle | 1.200 |
| Data reduction software | TRUNCATE |
| Data scaling software | CCP4 ((TRUNCATE)) |
| Phasing software | CNS |
| Refinement software | CNS |
Data quality characteristics
| Overall | Outer shell | |
| Low resolution limit [Å] | 45.970 | |
| High resolution limit [Å] | 2.380 | 2.380 |
| Rmerge | 0.087 | 0.333 |
| Number of reflections | 76808 | |
| <I/σ(I)> | 16.8 | 5.8 |
| Completeness [%] | 99.8 | 100 |
| Redundancy | 7.7 |
Crystallization Conditions
| crystal ID | method | pH | temperature | details |
| 1 | VAPOR DIFFUSION, HANGING DROP | 7.5 | 273 | A complex of 1x10^-4 M T7 DNA polymerase 5A7A:thioredoxin was assembled with an equimolar amount of double stranded DNA substrate. Crystallization was achieved using a buffer containing 50mM HEPES pH 7.5, 10mM MgCl_2, 2mM DTT, and 0.5 mM terminal ddTTP Seed crystals were grown by hanging drop vapor diffusion by mixing 1ul each of protein-DNA solution and a reservoir solutions containing between 16 to 20% PEG 8000, 100mM ACES pH 7.5, 120 ammonium sulfate, 30mM MgCl2, and 5mM DTT. These crystals were used to streak-seed a grid of protein/reservoir solutions with concentrations of PEG 8000 between 13 to 15%. Pyramidal crystals appeared overnight and reached a maximum size of ~150 X 150 X 100 um3 after 3 to 4 days. Crystals were harvested overnight in mother-liquor containing 10 % PEG 400, temperature 273K, VAPOR DIFFUSION, HANGING DROP |
Crystallization Reagents
| ID | crystal ID | solution ID | reagent name | concentration | details |
| 1 | 1 | 1 | HEPES | ||
| 10 | 1 | DTT | |||
| 11 | 1 | 2 | ddTTP | ||
| 12 | 1 | 2 | PEG 8000 | ||
| 13 | 1 | 2 | ACES | ||
| 14 | 1 | 2 | (NH4)2SO4 | ||
| 2 | 1 | 1 | MgCl2 | ||
| 3 | 1 | 1 | DTT | ||
| 4 | 1 | 1 | ddTTP | ||
| 5 | 1 | 1 | PEG 8000 | ||
| 6 | 1 | 1 | ACES | ||
| 7 | 1 | 1 | (NH4)2SO4 | ||
| 8 | 1 | 2 | HEPES | ||
| 9 | 1 | 2 | MgCl2 |
