Table 1.
Summary of data collection and processing parameters
| Parameter | 3KE mutant particle and substructure | Dimer particle and VPS35–VPS35 substructure | Heterotrimer particle and VPS35–VPS35 substructure | ||
|---|---|---|---|---|---|
| Microscope | Thermo Fisher FEI Titan Krios G3i Vanderbilt V-CEM | FEI Titan Krios Krios2 NRAMM Data collection 1 |
FEI Titan Krios Krios2 NRAMM Data collection 1 |
FEI Titan Krios Krios3 NRAMM Data collection 2 |
FEI Titan Krios Krios3 NRAMM Data collection 3 |
| Cs | 2.7 mm | 2.7 mm | 2.7 mm | 2.7 mm | 2.7 mm |
| Voltage | 300 keV | 300 keV | 300 keV | 300 keV | 300 keV |
| Detector | ThermoFisher Falcon 3 direct electron detector |
Gatan K2 Summit direct electron detector |
Gatan K2 Summit direct electron detector |
Gatan K2 Summit direct electron detector |
Gatan K2 Summit direct electron detector |
| Magnification | 120,000× | 105,000× | 105,000× | 105,000× | 105,000× |
| Pixel size | 0.6811 Å/pix | 1.096 Å/pix | 1.096 Å/pix | 1.06 Å/pix (rescaled to 1.096 Å/pix with motioncorr2 script) | 1.06 Å/pix (rescaled to 1.096 Å/pix with motioncorr2 script) |
| Dose rate | 1.4 e−/A2/s | ∼8 e−/Å2/s | ∼8 e−/Å2/s | ∼8 e−/Å2/s | ∼8 e−/Å2/s |
| Total dose | 50 e−/A2 | 69.34 e−/Å2 | 69.34 e−/Å2 | 73.92 e−/Å2 | 73.92 e−/Å2 |
| Tilt | ±30° | N/A | N/A | N/A | ±15° |
| Defocus range | −0.8 to −2.6 μm | −0.7 to 2.6 μm | −0.7 to 2.6 μm | −0.8 to −4.4 μm | −0.8 to −4.7 μm |
| Number of micrographs | 4791 | 1480 | 1480 | 1299 | 891 |
| Total particles (autopicked) | 275,633 | 533,231 | (250,500 particles selected from these datasets; (39)) | 207,026 | |
| Box size (Å) | 436 × 436 Å (3KE) 215 × 215 Å (substructure) |
351 × 351 Å (dimer) 180 × 180 Å (substructure) |
241 × 241 Å (heterotrimer) 180 × 180 Å (substructure) |
||
| Particles in 2D classification | 28,645 (3KE) 19,184 (substructure) |
88,113 (dimer) 70,214 (substructure) |
72,295 (heterotrimer) 69,381 (substructure) |
||
| Particles in final 3D model | 26,641 (3KE) 17,837 (substructure) |
70,214 (dimer) 17,840 (substructure) |
43,808 (heterotrimer) 69,381 (substructure) |
||
| Symmetry | C1 | C1 | C1 | ||
| Map resolution (masked FSC 0.143, RELION) | 7.1 Å (3KE) 7.2 Å (substructure) |
7.0 Å (dimer) 7.3 Å (substructure) |
4.9 Å (heterotrimer) 4.5 Å (substructure) |
||
| B-factor (RELION) | −226.1 (3KE) −244.6 (substructure) |
−227.0 (dimer) −150.1 (substructure) |
−113.8 (heterotrimer) −226.3 (substructure) |
||