Succinic semialdehyde dehydrogenase from E. coli with bound NAD+ and succinic semialdehyde
X-RAY DIFFRACTION
Crystallization
| Crystalization Experiments | ||||
|---|---|---|---|---|
| ID | Method | pH | Temperature | Details |
| 1 | VAPOR DIFFUSION, SITTING DROP | 295 | 7.3 mg/mL of SAD in 20 mM HEPES-KOH, 50 mM KCl, pH 7.5 was pre-mixed with NAD+ (final concentration 5 mM). The pre-mixed protein was then mixed in a 2:1 ratio with 250 mM Bis-Tris-Propane, pH 8.25, 20 % (w/v) PEG4000. The final drop size was 3 uL. The crystal was soaked with 5 mM succinic semialdehyde for 2 minutes and the drop was complemented with PEG200 to a final concentration of 20 % (v/v) before flash freezing the crystal in liquid nitrogen. | |
| Crystal Properties | |
|---|---|
| Matthews coefficient | Solvent content |
| 2.39 | 48.47 |
Crystal Data
| Unit Cell | |
|---|---|
| Length ( Å ) | Angle ( ˚ ) |
| a = 91.52 | α = 90 |
| b = 115.62 | β = 90 |
| c = 179.64 | γ = 90 |
| Symmetry | |
|---|---|
| Space Group | P 21 21 21 |
Diffraction
| Diffraction Experiment | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Crystal ID | Scattering Type | Data Collection Temperature | Detector | Detector Type | Details | Collection Date | Monochromator | Protocol | |||||
| 1 | 1 | x-ray | 100 | PIXEL | DECTRIS EIGER X 4M | 2023-05-18 | M | SINGLE WAVELENGTH | ||||||
| Radiation Source | |||||
|---|---|---|---|---|---|
| ID # | Source | Type | Wavelength List | Synchrotron Site | Beamline |
| 1 | SYNCHROTRON | ESRF BEAMLINE MASSIF-3 | 0.9677 | ESRF | MASSIF-3 |
Data Collection
| Overall | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Resolution (High) | Resolution (Low) | Percent Possible (Observed) | R Merge I (Observed) | Rrim I (All) | CC (Half) | 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 | 2.3 | 38.07 | 99.9 | 0.154 | 0.16399999999999998 | 0.998 | 9.59 | 8.4 | 85212 | ||||||||||
| Highest Resolution Shell | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Resolution (High) | Resolution (Low) | Percent Possible (All) | Percent Possible (Observed) | R Merge I (Observed) | Rrim I (All) | CC (Half) | Mean I Over Sigma (Observed) | Redundancy | Number Unique Reflections (All) | |||||||||
| 1 | 2.3 | 2.36 | 0.998 | 1.063 | 0.8590000000000001 | ||||||||||||||
Refinement
| Statistics | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Diffraction ID | Structure Solution Method | Cross Validation method | Resolution (High) | Resolution (Low) | Cut-off Sigma (F) | Number Reflections (Observed) | Number Reflections (R-Free) | Percent Reflections (Observed) | R-Factor (Observed) | R-Work (Depositor) | R-Work (DCC) | R-Free (Depositor) | R-Free (DCC) | Mean Isotropic B | |||||
| X-RAY DIFFRACTION | MOLECULAR REPLACEMENT | FREE R-VALUE | 2.3 | 38.07 | 1.36 | 85024 | 1995 | 99.7 | 0.197 | 0.1962 | 0.195 | 0.2278 | 0.2263 | ||||||
| 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] | |
| RMS Deviations | |
|---|---|
| Key | Refinement Restraint Deviation |
| f_dihedral_angle_d | 11.968 |
| f_angle_d | 0.54 |
| f_chiral_restr | 0.041 |
| f_plane_restr | 0.004 |
| f_bond_d | 0.003 |
| Non-Hydrogen Atoms Used in Refinement | |
|---|---|
| Non-Hydrogen Atoms | Number |
| Protein Atoms | 13904 |
| Nucleic Acid Atoms | |
| Solvent Atoms | 461 |
| Heterogen Atoms | 204 |
Software
| Software | |
|---|---|
| Software Name | Purpose |
| PHENIX | refinement |
| XSCALE | data scaling |
| XDS | data reduction |
| PHENIX | phasing |
