Succinic semialdehyde dehydrogenase from E. coli with Q262R substitution and bound NAD+
X-RAY DIFFRACTION
Crystallization
| Crystalization Experiments | ||||
|---|---|---|---|---|
| ID | Method | pH | Temperature | Details |
| 1 | VAPOR DIFFUSION, SITTING DROP | 8.25 | 295 | 35.0 mg/mL of SAD_Q262R in 20 mM HEPES-KOH, 50 mM KCl, pH 7.5 was supplemented with 5 mM NAD+ and 1 mM TCEP. The enzyme was then mixed in a 1:1 ratio with 285 mM Bis-Tris-Propane, 8.25 pH , 17 % (w/v) PEG4000. The final drop size was 4 uL. The drop was complemented with PEG200 to a final concentration of 16 % (v/v) before flash freezing the crystals in liquid nitrogen. |
| Crystal Properties | |
|---|---|
| Matthews coefficient | Solvent content |
| 2.38 | 48.34 |
Crystal Data
| Unit Cell | |
|---|---|
| Length ( Å ) | Angle ( ˚ ) |
| a = 91.3 | α = 90 |
| b = 115.69 | β = 90 |
| c = 179.61 | 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 EIGER2 X CdTe 16M | 2023-03-23 | M | SINGLE WAVELENGTH | ||||||
| Radiation Source | |||||
|---|---|---|---|---|---|
| ID # | Source | Type | Wavelength List | Synchrotron Site | Beamline |
| 1 | SYNCHROTRON | PETRA III, EMBL c/o DESY BEAMLINE P14 (MX2) | 0.9763 | PETRA III, EMBL c/o DESY | P14 (MX2) |
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 | 1.9 | 29.46 | 99.5 | 0.133 | 0.138 | 0.9990000000000001 | 13.34 | 13.8 | 149467 | ||||||||||
| 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 | 1.9 | 1.95 | 0.998 | 1.036 | 0.92 | ||||||||||||||
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 | 1.9 | 29.46 | 1.36 | 149117 | 1996 | 99.32 | 0.1818 | 0.1813 | 0.181 | 0.2133 | 0.2128 | ||||||
| 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 | 12.505 |
| f_angle_d | 0.728 |
| f_chiral_restr | 0.046 |
| f_plane_restr | 0.006 |
| f_bond_d | 0.004 |
| Non-Hydrogen Atoms Used in Refinement | |
|---|---|
| Non-Hydrogen Atoms | Number |
| Protein Atoms | 13912 |
| Nucleic Acid Atoms | |
| Solvent Atoms | 1129 |
| Heterogen Atoms | 176 |
Software
| Software | |
|---|---|
| Software Name | Purpose |
| PHENIX | refinement |
| XSCALE | data scaling |
| XDS | data reduction |
| PHENIX | phasing |
