3OWV
Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae
Experimental procedure
| Experimental method | SINGLE WAVELENGTH |
| Source type | SYNCHROTRON |
| Source details | APS BEAMLINE 22-ID |
| Synchrotron site | APS |
| Beamline | 22-ID |
| Temperature [K] | 93 |
| Detector technology | CCD |
| Collection date | 2009-12-10 |
| Detector | MARMOSAIC 225 mm CCD |
| Wavelength(s) | 0.94 |
| Spacegroup name | P 21 21 21 |
| Unit cell lengths | 67.963, 75.931, 90.784 |
| Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
| Resolution | 37.970 - 1.750 |
| R-factor | 0.195 |
| Rwork | 0.195 |
| R-free | 0.21700 |
| Structure solution method | MOLECULAR REPLACEMENT |
| Starting model (for MR) | incomplete manually built model of EndA from a low resolution incompletely refined data set from an MBP-EndA fusion protein |
| RMSD bond length | 0.004 |
| RMSD bond angle | 1.200 |
| Data reduction software | HKL-2000 |
| Data scaling software | HKL-2000 |
| Phasing software | AutoBuild |
| Refinement software | CNS (1.2) |
Data quality characteristics
| Overall | Outer shell | |
| Low resolution limit [Å] | 50.000 | 1.780 |
| High resolution limit [Å] | 1.750 | 1.750 |
| Number of reflections | 46402 | |
| <I/σ(I)> | 26.5 | 3.56 |
| Completeness [%] | 96.8 | 68.7 |
| Redundancy | 13.6 | 7.2 |
Crystallization Conditions
| crystal ID | method | pH | temperature | details |
| 1 | VAPOR DIFFUSION, SITTING DROP | 7 | 297 | Crystallization solution contained 0.1M Tris pH 7, 17% PEG8000, and 0.2M MgCl2. The crystal was transferred to a hanging drop containing 0.1M Tris pH 7, 75mM MaCl, 0.2M MgCl2, and 20% PEG800, which was then placed over a vapor diffusion chamber containing a sitting drop bridge with 25% glutaraldehyde. The crystal was cross-linked for 8 minutes at room temperature, then transferred to a soaking solution containing 0.1M Tris pH 7, 75mM NaCl, 0.2M MgCl2,20% PEG8000, 11.6mM trimethyl lead acetate, and 9.63mM triethyl lead acetate for 43 hrs at room temperature. The soaked crystal was then transferred to a cryoprotectant (0.1M Tris pH 7, 75mM NaCl, 0.2M MgCl2, 20% PEG8000, 15% ethylene glycol, 11.9mM trimethyl lead acetate, and 13.75mM triethyl lead acetate) in four steps, then flash frozen in liquid nitrogen. , VAPOR DIFFUSION, SITTING DROP, temperature 297K |
