6I7T
eIF2B:eIF2 complex
ELECTRON MICROSCOPY
| Sample |
|---|
| Complex of translation initiation factors eIF2 and eIF2B |
| Specimen Preparation | |
|---|---|
| Sample Aggregation State | PARTICLE |
| Vitrification Instrument | FEI VITROBOT MARK III |
| Cryogen Name | ETHANE |
| Sample Vitrification Details | Blot for 1 second before plunging |
| 3D Reconstruction | |
|---|---|
| Reconstruction Method | SINGLE PARTICLE |
| Number of Particles | 23274 |
| Reported Resolution (Å) | 4.61 |
| Resolution Method | FSC 0.143 CUT-OFF |
| Other Details | |
| Refinement Type | |
| Symmetry Type | POINT |
| Point Symmetry | C2 |
| Map-Model Fitting and Refinement | |||||
|---|---|---|---|---|---|
| Id | 1 (6I3M, 6I3M, 6I3M, 6I3M, 6I3M, 6I3M, 6I3M, 6I3M, 6I3M, 6I3M, 6I3M, 6I3M) | 2 (3JAP, 3JAP, 3JAP) | |||
| Refinement Space | REAL | REAL | |||
| Refinement Protocol | FLEXIBLE FIT | RIGID BODY FIT | |||
| Refinement Target | correlation coeficient | Cross-correlation coefficient | |||
| Overall B Value | |||||
| Fitting Procedure | |||||
| Details | Our model of eIF2aP:eIF2B (PDB code:6I3M) was fitted and then iteratively refined using phenix and manually adjusted in Coot. | eIF2 alpha domain 3, eIF2 gamma, and eIF2 beta (3JAP) were rigid body fitted into our map, Fourier filtered 15A resolution, using UCSF_Chimera. | |||
| Data Acquisition | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Detector Type | GATAN K2 QUANTUM (4k x 4k) | ||||||||
| Electron Dose (electrons/Å**2) | 1 | ||||||||
| Imaging Experiment | 1 |
|---|---|
| Date of Experiment | |
| Temperature (Kelvin) | |
| Microscope Model | FEI TITAN KRIOS |
| Minimum Defocus (nm) | 1000 |
| Maximum Defocus (nm) | 3000 |
| Minimum Tilt Angle (degrees) | |
| Maximum Tilt Angle (degrees) | |
| Nominal CS | 2.7 |
| Imaging Mode | BRIGHT FIELD |
| Specimen Holder Model | FEI TITAN KRIOS AUTOGRID HOLDER |
| Nominal Magnification | |
| Calibrated Magnification | 37313 |
| Source | FIELD EMISSION GUN |
| Acceleration Voltage (kV) | 300 |
| Imaging Details | Tilt at 35 degrees. |
| EM Software | ||
|---|---|---|
| Task | Software Package | Version |
| PARTICLE SELECTION | RELION | 2.1 |
| IMAGE ACQUISITION | EPU | |
| CTF CORRECTION | Gctf | |
| MODEL FITTING | UCSF Chimera | |
| MODEL FITTING | Coot | |
| INITIAL EULER ASSIGNMENT | RELION | 2.1 |
| FINAL EULER ASSIGNMENT | RELION | 2.1 |
| CLASSIFICATION | RELION | 2.1 |
| RECONSTRUCTION | RELION | 2.1 |
| MODEL REFINEMENT | PHENIX | |
| MODEL FITTING | UCSF Chimera | |
| Image Processing | ||||
|---|---|---|---|---|
| CTF Correction Type | CTF Correction Details | Number of Particles Selected | Particle Selection Details | |
| PHASE FLIPPING AND AMPLITUDE CORRECTION | CTF determination was performed per micrograph initially. After particle picking and initial reconstruction, per particle CTF determination was performed. | 114390 | 2024 images were selected for particle picking. Initially, 3090 particles were picked manually and used for 2D classification into 20 classes. The best 8 classes (containing 2162 particles) were selected and used as references for automated particle picking in RELION. Particles on the lacey carbon, not in holes, were manually deleted in RELION, before extraction and further iterative cleaning by classification. | |
