4X6R
An Isoform-specific Myristylation Switch Targets RIIb PKA Holoenzymes to Membranes
X-RAY DIFFRACTION
Crystallization
| Crystalization Experiments | ||||
|---|---|---|---|---|
| ID | Method | pH | Temperature | Details |
| 1 | BATCH MODE | 298 | Crystallization of RIb(91-379,R333K): myrC(K7C) heterodimer :RC heterodimer that was concentrated to 14 mg/mL and screened against different ammonium sulfate concentrations ranging from 0.8-2.5 M in 0.1 M sodium citrate buffer and also varying the pH from 5.0-6.0 using the hanging drop vapor diffusion method. The crystal used for structure determination was obtained from a 4 uL drop containing 1:1 protein to well solution with the well solution containing 1.6 M ammonium sulfate and 0.1 M sodium citrate at pH 5.5 | |
| Crystal Properties | |
|---|---|
| Matthews coefficient | Solvent content |
| 4.3 | 71.41 |
Crystal Data
| Unit Cell | |
|---|---|
| Length ( Å ) | Angle ( ˚ ) |
| a = 125.76 | α = 90 |
| b = 125.76 | β = 90 |
| c = 140.881 | γ = 120 |
| Symmetry | |
|---|---|
| Space Group | P 32 2 1 |
Diffraction
| Diffraction Experiment | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Crystal ID | Scattering Type | Data Collection Temperature | Detector | Detector Type | Details | Collection Date | Monochromator | Protocol | |||||
| 1 | 1 | x-ray | 200 | CCD | ADSC QUANTUM 1 | 2013-07-04 | M | SINGLE WAVELENGTH | ||||||
| Radiation Source | |||||
|---|---|---|---|---|---|
| ID # | Source | Type | Wavelength List | Synchrotron Site | Beamline |
| 1 | SYNCHROTRON | ALS BEAMLINE 8.2.1 | 1 | ALS | 8.2.1 |
Data Collection
| Overall | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Resolution (High) | Resolution (Low) | Percent Possible (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 | 2.4 | 63 | 99.9 | 3 | 4 | 48209 | |||||||||||||
Refinement
| Statistics | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Diffraction ID | Structure Solution Method | Cross Validation method | Resolution (High) | Resolution (Low) | Cut-off Sigma (F) | Number Reflections (R-Free) | Percent Reflections (Observed) | R-Factor (Observed) | R-Work | R-Free | Mean Isotropic B | ||||||||
| X-RAY DIFFRACTION | FREE R-VALUE | 2.4 | 62.88 | 1.6 | 2575 | 99.9 | 0.186 | 0.184 | 0.234 | ||||||||||
| 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] | |
| 1.8891 | 1.8891 | -3.7782 | ||||
| RMS Deviations | |
|---|---|
| Key | Refinement Restraint Deviation |
| f_dihedral_angle_d | 17.822 |
| f_angle_d | 1.857 |
| f_chiral_restr | 0.133 |
| f_bond_d | 0.022 |
| f_plane_restr | 0.01 |
| Non-Hydrogen Atoms Used in Refinement | |
|---|---|
| Non-Hydrogen Atoms | Number |
| Protein Atoms | 5145 |
| Nucleic Acid Atoms | |
| Solvent Atoms | 269 |
| Heterogen Atoms | 101 |
Software
| Software | |
|---|---|
| Software Name | Purpose |
| PHENIX | refinement |
| MOSFLM | data reduction |
| HKL-2000 | data scaling |
| PHASER | phasing |
