Table 1. Cryo-EM data collection, refinement and validation statistics.
| Map A (EMDB-11673) Apo Pol III (PDB 7A6H) |
Map B (EMDB-11736) EC-1 Pol III (PDB 7AE1) | Map C (EMDB-11737) | Map D (EMDB-11738) EC-3 Pol III (PDB 7AE3) | |
|---|---|---|---|---|
| Data collection and processing | ||||
| Magnification | 105,000 | 130,000 | 130,000 | 130,000 |
| Voltage (kV) | 300 | 300 | 300 | 300 |
| Electron exposure (e–/Å2) | 38.11 | 40.35 | 40.35 | 40.35 |
| Defocus range (μm) | 0.75-2.25 | 0.75-2.00 | 0.75-2.00 | 0.75-2.00 |
| Pixel size (Å) | 0.822 | 1.05 | 1.05 | 1.05 |
| Symmetry imposed | C1 | C1 | C1 | C1 |
| Initial particle images (no.) | 304,683 | 367,717 | 367,717 | 367,717 |
| Final particle images (no.) | 111,289 | 166,071 | 70,492 | 30,525 |
| Map resolution (Å) | 3.3 | 2.8 | 3.1 | 3.1 |
| FSC threshold | 0.143 | 0.143 | 0.143 | 0.143 |
| Map resolution range (Å) | 3.0 – 8.3 | 2.5 – 7.8 | 2.8 – 8.3 | 2.9 – 6.9 |
| Refinement | ||||
| Initial model used (PDB code) | 3AYH,5AFQ,5F J8,5FJ9, 5FLM,5IY6, 6CND,6EU0, 6EU1,6EU2 |
3AYH,5AFQ,5F J8,5FJ9, 5FLM,5IY6, 6CND,6EU0, 6EU1,6EU2 |
3AYH,5AFQ,5F J8,5FJ9, 5FLM,5IY6, 6CND,6EU0, 6EU1,6EU2 |
|
| Model resolution (Å) | 3.4 | 3.0 | 3.2 | |
| Map sharpening B factor (Å2) | -80.1 | -55.2 | -49.6 | |
| Model composition | ||||
| Non-hydrogen atoms | 40217 | 42609 | 42526 | |
| Protein / Nucleotide residues | 5072 / - | 5258 / 44 | 5247 / 44 | |
| Ligands | 1x SF4, 7x Zn, 1x Mg | 1x SF4, 7x Zn, 1x Mg | 1x SF4, 7x Zn, 1x Mg | |
| B factors (Å2) | ||||
| Protein | 105.65 | 84.75 | 126.86 | |
| Ligand | 105.80 | 93.50 | 132.15 | |
| Nucleotide | 155.90 | 180.37 | ||
| R.m.s. deviations | ||||
| Bond lengths (Å) | 0.010 | 0.008 | 0.008 | |
| Bond angles (°) | 1.271 | 1.315 | 1.208 | |
| Validation | ||||
| MolProbity score | 1.54 | 1.28 | 1.43 | |
| Clashscore | 3.91 | 2.20 | 3.46 | |
| Poor rotamers (%) | 0.25 | 0.43 | 0.32 | |
| Ramachandran plot | ||||
| Favored (%) | 94.71 | 95.90 | 95.71 | |
| Allowed (%) | 5.23 | 3.99 | 4.23 | |
| Disallowed (%) | 0.06 | 0.12 | 0.06 | |
| Map E (EMDB-11739) | Map F (EMDB-11740) | Map G1 (EMDB-11741) | Map H1 (EMDB-11742) EC-2 Pol III (PDB 7AEA) | |
|---|---|---|---|---|
| Data collection and processing | ||||
| Magnification | 130,000 | 130,000 | 105,000/130,000 | 105,000/130,000 |
| Voltage (kV) | 300 | 300 | 300 | 300 |
| Electron exposure (e–/Å2) | 40.35 | 40.35 | 38.11/40.35 | 38.11/40.35 |
| Defocus range (μm) | 0.75-2.00 | 0.75-2.00 | 0.75-2.25/0.75-2.00 | 0.75-2.25/0.75-2.00 |
| Pixel size (Å) | 1.05 | 1.05 | 1.05 | 1.05 |
| Symmetry imposed | C1 | C1 | C1 | C1 |
| Initial particle images (no.) | 367,717 | 367,717 | 277,360 | 277,360 |
| Final particle images (no.) | 100,152 | 21,861 | 277,360 | 31,737 |
| Map resolution (Å) | 2.9 | 3.3 | 2.9 | 3.3 |
| FSC threshold | 0.143 | 0.143 | 0.143 | 0.143 |
| Map resolution range (Å) | 2.6 – 7.6 | 3.0 – 8.9 | 2.8 – 7.1 | 3.2 – 10.0 |
| Refinement | ||||
| Initial model used (PDB code) | 3AYH,5AFQ,5F J8,5FJ9, 5FLM,5IY6, 6CND,6EU0, 6EU1,6EU2 |
|||
| Model resolution (Å) | 3.4 | |||
| Map sharpening B factor (Å2) | -43.6 | |||
| Model composition | ||||
| Non-hydrogen atoms | 42423 | |||
| Protein / Nucleotide residues | 5218 / 44 | |||
| Ligands | 1x SF4, 7x Zn, 1x Mg | |||
| B factors (Å2) | ||||
| Protein | 117.34 | |||
| Ligand | 143.32 | |||
| Nucleotide | 222.81 | |||
| R.m.s. deviations | ||||
| Bond lengths (Å) | 0.006 | |||
| Bond angles (°) | 1.156 | |||
| Validation | ||||
| MolProbity score | 1.44 | |||
| Clashscore | 3.48 | |||
| Poor rotamers (%) | 0.28 | |||
| Ramachandran plot | ||||
| Favored (%) | 95.64 | |||
| Allowed (%) | 4.30 | |||
| Disallowed (%) | 0.06 | |||
1
Map G and map H derived from a combined data set of apo and elongating Pol III by merging final particle images of map A and map B. Data collection and processing values for both data sets are given.