ELECTRON MICROSCOPY
Starting Model(s)
| Initial Refinement Model(s) | |||
|---|---|---|---|
| Type | Source | Accession Code | Details |
| experimental model | PDB | 3JAR | |
| Sample |
|---|
| Deacetylated Microtubule |
| Specimen Preparation | |
|---|---|
| Sample Aggregation State | HELICAL ARRAY |
| Vitrification Instrument | FEI VITROBOT MARK IV |
| Cryogen Name | ETHANE |
| Sample Vitrification Details | Blotted for 4 seconds at blot force 10. |
| 3D Reconstruction | |
|---|---|
| Reconstruction Method | HELICAL |
| Number of Particles | 24692 |
| Reported Resolution (Å) | 4 |
| Resolution Method | FSC 0.143 CUT-OFF |
| Other Details | |
| Refinement Type | |
| Symmetry Type | HELICAL |
| Axial Symmetry | C1 |
| Axial Rise | 9.3 |
| Angular Rotation | -27.7 |
| Map-Model Fitting and Refinement | |||||
|---|---|---|---|---|---|
| Id | 1 (3JAR) | ||||
| Refinement Space | REAL | ||||
| Refinement Protocol | BACKBONE TRACE | ||||
| Refinement Target | 0.5 | ||||
| Overall B Value | 126 | ||||
| Fitting Procedure | |||||
| Details | We use PHENIX to perform real space refinement and sharpen our cryoEM maps. | ||||
| Data Acquisition | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Detector Type | GATAN K2 SUMMIT (4k x 4k) | ||||||||
| Electron Dose (electrons/Å**2) | 25 | ||||||||
| Imaging Experiment | 1 |
|---|---|
| Date of Experiment | |
| Temperature (Kelvin) | |
| Microscope Model | FEI TITAN KRIOS |
| Minimum Defocus (nm) | 1500 |
| Maximum Defocus (nm) | 2500 |
| 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 | 22500 |
| Calibrated Magnification | 23364 |
| Source | FIELD EMISSION GUN |
| Acceleration Voltage (kV) | 300 |
| Imaging Details | Preliminary grid screening was performed manually and all of the alignments were initially done using a gold calibration grid. |
| EM Software | ||
|---|---|---|
| Task | Software Package | Version |
| PARTICLE SELECTION | Appion | 2.0 |
| IMAGE ACQUISITION | SerialEM | 3.7 |
| CTF CORRECTION | CTFFIND | 4 |
| MODEL FITTING | PHENIX | 1.14 |
| MODEL REFINEMENT | PHENIX | 1.14 |
| INITIAL EULER ASSIGNMENT | EMAN | 1.9 |
| FINAL EULER ASSIGNMENT | EMAN | 1.9 |
| CLASSIFICATION | EMAN | 1.9 |
| RECONSTRUCTION | FREALIGN | 9.11 |
| Image Processing | ||||
|---|---|---|---|---|
| CTF Correction Type | CTF Correction Details | Number of Particles Selected | Particle Selection Details | |
| PHASE FLIPPING AND AMPLITUDE CORRECTION | CTFFIND4 | 29396 | Extracted Helical Segments | |
