5MQ0
Structure of a spliceosome remodeled for exon ligation
ELECTRON MICROSCOPY
| Sample |
|---|
| Saccharomyces cerevisiae spliceosome. Complex C just after Prp16-mediated remodeling |
| Specimen Preparation | |
|---|---|
| Sample Aggregation State | PARTICLE |
| Vitrification Instrument | FEI VITROBOT MARK III |
| Cryogen Name | ETHANE |
| Sample Vitrification Details | 3.5 microlitres sample were applied to the grid, left for 25 seconds and then blotted for 3.0-3.5 seconds before plunging. |
| 3D Reconstruction | |
|---|---|
| Reconstruction Method | SINGLE PARTICLE |
| Number of Particles | 29527 |
| Reported Resolution (Å) | 4.17 |
| Resolution Method | FSC 0.143 CUT-OFF |
| Other Details | A 3D reconstruction was obtained after refinement of a subset of particles obtained by classification with a mask around Prp22. |
| Refinement Type | |
| Symmetry Type | POINT |
| Point Symmetry | C1 |
| Map-Model Fitting and Refinement | |||||
|---|---|---|---|---|---|
| Id | 1 | ||||
| Refinement Space | RECIPROCAL | ||||
| Refinement Protocol | FLEXIBLE FIT | ||||
| Refinement Target | Fourier Shell Correlation | ||||
| Overall B Value | 330 | ||||
| Fitting Procedure | |||||
| Details | Used secondary structure restraints generated in ProSMART and LibG. | ||||
| Data Acquisition | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Detector Type | GATAN K2 SUMMIT (4k x 4k) | ||||||||
| Electron Dose (electrons/Å**2) | 2 | ||||||||
| Imaging Experiment | 1 |
|---|---|
| Date of Experiment | |
| Temperature (Kelvin) | |
| Microscope Model | FEI TITAN KRIOS |
| Minimum Defocus (nm) | 500 |
| Maximum Defocus (nm) | 4500 |
| Minimum Tilt Angle (degrees) | |
| Maximum Tilt Angle (degrees) | |
| Nominal CS | |
| Imaging Mode | BRIGHT FIELD |
| Specimen Holder Model | FEI TITAN KRIOS AUTOGRID HOLDER |
| Nominal Magnification | 81000 |
| Calibrated Magnification | |
| Source | FIELD EMISSION GUN |
| Acceleration Voltage (kV) | 300 |
| Imaging Details | GIF Quantum energy filter, 20 eV slit width |
| EM Software | ||
|---|---|---|
| Task | Software Package | Version |
| PARTICLE SELECTION | RELION | 1.4 |
| CTF CORRECTION | CTFFIND | 4 |
| MODEL FITTING | Coot | 0.8.3 |
| MODEL REFINEMENT | REFMAC | 5.8 |
| INITIAL EULER ASSIGNMENT | RELION | 1.4 |
| FINAL EULER ASSIGNMENT | RELION | 1.4 |
| CLASSIFICATION | RELION | 1.4 |
| RECONSTRUCTION | RELION | 1.4 |
| Image Processing | ||||
|---|---|---|---|---|
| CTF Correction Type | CTF Correction Details | Number of Particles Selected | Particle Selection Details | |
| PHASE FLIPPING AND AMPLITUDE CORRECTION | 350000 | Selected initial particles automatically using C complex 2D class averages, low-pass filtered to 20 Angstrom for automatic particle picking. | ||
