4G29
Structure of the Catalytic Domain of the Salmonella Virulence Factor SseI
Experimental procedure
| Experimental method | SINGLE WAVELENGTH |
| Source type | SYNCHROTRON |
| Source details | NSLS BEAMLINE X29A |
| Synchrotron site | NSLS |
| Beamline | X29A |
| Temperature [K] | 93 |
| Detector technology | CCD |
| Detector | ADSC QUANTUM 315 |
| Wavelength(s) | 0.97900 |
| Spacegroup name | P 41 21 2 |
| Unit cell lengths | 52.256, 52.256, 132.146 |
| Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
| Resolution | 19.080 - 1.700 |
| R-factor | 0.18469 |
| Rwork | 0.183 |
| R-free | 0.21364 |
| Structure solution method | SAD |
| RMSD bond length | 0.019 |
| RMSD bond angle | 1.647 |
| Data reduction software | HKL-2000 |
| Data scaling software | HKL-2000 |
| Refinement software | REFMAC (5.5.0044) |
Data quality characteristics
| Overall | |
| High resolution limit [Å] | 1.700 |
| Number of reflections | 19151 |
Crystallization Conditions
| crystal ID | method | pH | temperature | details |
| 1 | VAPOR DIFFUSION, HANGING DROP | 4.6 | 277.15 | Crystallization screens were conducted using 1:1 sitting drops at RT and 4 C. The C-terminal domain (137-322) (~20mg/mL) gave large rectangular crystals in 1.15M LiSO4, 0.1M NaOAc, pH 4.6 (4 C) when optimized (1:1 hanging drops). However these were very fragile and consisted of multiple lattices overlaid on each other as observed in their diffraction patterns. SseI 137-322 was then reductively methylated and rescreened. It gave multiple crystals of differing morphologies in a number of conditions containing MES, Tris, and HEPES buffers pH 6.0 - 9.0, and 1.6 to 2.0M (NH4)SO4. To obtain single crystals, crystals from 0.1M MES pH 7.1, 1.8M (NH4)SO4 were crushed and microseeded into pre-equilibrated (2 hrs) drops containing protein/reservoir solution (0.1M MES pH 5.5, 1.6M (NH4)SO4). This procedure produced single, small, rectangular crystals. To obtain larger crystals, single crystals obtained in the previous step were macroseeded into pre-equilibrated drops, giving large, thin rectangular plates that were well-diffracting. SeMet-substituted crystals of 137-322 were obtained in the same conditions but gave large crystals directly without any seeding. Crystals were then cryoprotected by transfer in small increments into mother liquor supplemented with 25% glycerol., VAPOR DIFFUSION, HANGING DROP, temperature 277.15K |
