3IEC
Helicobacter pylori CagA Inhibits PAR1/MARK Family Kinases by Mimicking Host Substrates
X-RAY DIFFRACTION
Crystallization
| Crystalization Experiments | ||||
|---|---|---|---|---|
| ID | Method | pH | Temperature | Details |
| 1 | VAPOR DIFFUSION, HANGING DROP | 296 | Initial crystals were grown by vapor diffusion using hanging drops formed from mixing a 2:1 volume ratio of the protein complex with an equilibration buffer consisting of 22.5-25% polyethylene glycol (PEG) molecular weight 3350 Da, 300 mM Li2SO4, and 100 mM Bis-Tris pH 5.8-6.2, at 23 C. Higher quality crystals were obtained by micro-seeding and addition of 5% PEG 400 as an additive. For cryoprotection crystals where transferred directly into a buffer with a 25% PEG 3350 Da, 5-10% PEG 400 Da, 300mM Li2SO4, 100mM Bis-Tris pH 5.8-6.2, 7.5% glycerol, and flash-cooled immediately afterward to -160 C. , VAPOR DIFFUSION, HANGING DROP, temperature 296K | |
| Crystal Properties | |
|---|---|
| Matthews coefficient | Solvent content |
| 2.42 | 49.2 |
Crystal Data
| Unit Cell | |
|---|---|
| Length ( Å ) | Angle ( ˚ ) |
| a = 93.466 | α = 90 |
| b = 93.256 | β = 100.94 |
| c = 113.955 | γ = 90 |
| Symmetry | |
|---|---|
| Space Group | P 1 21 1 |
Diffraction
| Diffraction Experiment | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Crystal ID | Scattering Type | Data Collection Temperature | Detector | Detector Type | Details | Collection Date | Monochromator | Protocol | |||||
| 1 | 1 | x-ray | 103 | CCD | ADSC QUANTUM 315r | Beamline X29 features high-flux radiation derived from a 54-pole harmonic emission undulator, a vertically focusing mirror and a horizontally focusing monochromator. A Cryomech AL300 is utilized to produce extremely stable cooling on a Si(111) crystal. The beamline is capable of delivering an energy range of 7-15 kev | 2008-08-08 | M | SINGLE WAVELENGTH | |||||
| Radiation Source | |||||
|---|---|---|---|---|---|
| ID # | Source | Type | Wavelength List | Synchrotron Site | Beamline |
| 1 | SYNCHROTRON | NSLS BEAMLINE X29A | 1.0809 | NSLS | X29A |
Data Collection
| Overall | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Resolution (High) | Resolution (Low) | Percent Possible (Observed) | R Merge I (Observed) | Net I Over Average Sigma (I) | Redundancy | Number Reflections (All) | Number Reflections (Observed) | Observed Criterion Sigma (F) | Observed Criterion Sigma (I) | B (Isotropic) From Wilson Plot | ||||||||
| 1 | 2.2 | 50 | 99.9 | 0.089 | 10.5 | 6.2 | 97843 | ||||||||||||
| Highest Resolution Shell | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Resolution (High) | Resolution (Low) | Percent Possible (All) | Percent Possible (Observed) | R Merge I (Observed) | Mean I Over Sigma (Observed) | Redundancy | Number Unique Reflections (All) | |||||||||||
| 1 | 2.2 | 2.28 | 100 | 0.706 | 6.2 | 9784 | |||||||||||||
Refinement
| Statistics | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Diffraction ID | Structure Solution Method | Cross Validation method | Resolution (High) | Resolution (Low) | Number Reflections (Observed) | Number Reflections (R-Free) | Percent Reflections (Observed) | R-Factor (Observed) | R-Work | R-Free | R-Free Selection Details | Mean Isotropic B | |||||||
| X-RAY DIFFRACTION | MOLECULAR REPLACEMENT | THROUGHOUT | 2.2 | 40.96 | 97422 | 4890 | 99.84 | 0.206 | 0.204 | 0.248 | RANDOM | 26.151 | |||||||
| Temperature Factor Modeling | ||||||
|---|---|---|---|---|---|---|
| Anisotropic B[1][1] | Anisotropic B[1][2] | Anisotropic B[1][3] | Anisotropic B[2][2] | Anisotropic B[2][3] | Anisotropic B[3][3] | |
| 0.28 | 0.28 | -0.38 | 0.21 | |||
| RMS Deviations | |
|---|---|
| Key | Refinement Restraint Deviation |
| r_dihedral_angle_2_deg | 32.815 |
| r_dihedral_angle_4_deg | 18.5 |
| r_dihedral_angle_3_deg | 17.087 |
| r_dihedral_angle_1_deg | 6.237 |
| r_scangle_it | 3.533 |
| r_scbond_it | 2.23 |
| r_angle_refined_deg | 1.507 |
| r_mcangle_it | 1.442 |
| r_angle_other_deg | 0.914 |
| r_mcbond_it | 0.768 |
| Non-Hydrogen Atoms Used in Refinement | |
|---|---|
| Non-Hydrogen Atoms | Number |
| Protein Atoms | 10625 |
| Nucleic Acid Atoms | |
| Solvent Atoms | 687 |
| Heterogen Atoms | |
Software
| Software | |
|---|---|
| Software Name | Purpose |
| DENZO | data reduction |
| SCALEPACK | data scaling |
| PHASER | phasing |
| REFMAC | refinement |
| PDB_EXTRACT | data extraction |
| HKL-2000 | data collection |
