Structure of catalase determined by MicroED
- Janelia Research Campus, Howard Hughes Medical Institute, United States
Figures
Figure 1
Diffraction from catalase microcrystals.
Representative untilted still diffraction pattern of a catalase microcrystal that shows sharp reflections extending to approximately 3.0 Å. Crystals of this quality were used to collect a data set …
Figure 2 with 1 supplement
Final structure of catalase at 3.2 Å resolution determined by MircoED.
(A) The complete refined catalase structure shown as ribbons. The corresponding 2mFobs-DFcalc density map around the complete structure can be seen in Video 2. (B) The density map, contoured at 1.5 …
Figure 2—figure supplement 1
Molecular replacement validation tests.
(A) The results of the single monomer MR show that all four chains of the tetramer could be properly placed. The RMSD between this MR solution and the original MR solution from complete tetramer …
Figure 3
Final Model validation tests.
(A–D) To check for model bias in the final MicroED data (A and C), and to compare with data obtained from synchrotron X-ray diffraction (B and D), residues 181–185 (A–B) or the heme groups (C–D) …
Videos
Video 1
Catalase diffraction data set collected by continuous rotation.
Diffraction data was recorded at an exposure time of 6 s per frame from a single crystal as the stage was continuously rotating at ∼0.09° s−1.
Video 2
Final density map and model of catalase at 3.2 Å.
The 2mFobs-DFcalc density map contoured at 1.5 σ shows good agreement with the final refined model.
Tables
Table 1
Resolution and completeness for catalase data sets
| Crystal 1 | Crystal 2 | Crystal 3 | Crystal 4* | Crystal 5 | |
|---|---|---|---|---|---|
| Resolution (Å) | 27.9–2.8 | 21.1–2.9 | 16.4–2.9 | 21.2–3.0 | 20.8–3.5 |
| (3.0–2.8) | (3.0–2.9) | (3.0–2.9) | (3.1–3.0) | (3.9–3.5) | |
| Unit cell dimensions | |||||
| a (Å) | 68.7 | 68.5 | 67.8 | 67.8 | 67.9 |
| b (Å) | 170.4 | 170.1 | 171.1 | 172.1 | 171.3 |
| c (Å) | 205.0 | 203.6 | 204.5 | 182.1 | 202.3 |
| α = β = γ (°) | 90 | 90 | 90 | 90 | 90 |
| Completeness (%) | 30.2 | 28.1 | 51.7 | 76.5 | 61.6 |
| (37.1) | (24.0) | (42.2) | (62.4) | (56.9) | |
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Values in parentheses reflect the highest resolution shell.
-
*
Data set used for final structure.
Table 2
Comparison of merging and refinement statistics for catalase data sets
| Multi-2.8 Å | Multi-3.0 Å | Multi-3.2 Å | Single-3.0 Å | Single-3.2 Å* | |
|---|---|---|---|---|---|
| Total crystals | 5 | 5 | 5 | 1 | 1 |
| Resolution (Å) | 27.8–2.8 | 27.8–3.0 | 27.8–3.2 | 21.2–3.0 | 21.2–3.2 |
| (2.9–2.8) | (3.1–3.0) | (3.4–3.2) | (3.1–3.0) | (3.4–3.2) | |
| Rmerge (%) | 25.8 | 25.5 | 24.8 | 18.7 | 17.5 |
| (42.0) | (41.2) | (38.2) | (38.8) | (32.7) | |
| CC1/2 | 0.906 | 0.920 | 0.933 | 0.886 | 0.891 |
| (0.231) | (0.348) | (0.574) | (0.426) | (0.555) | |
| Multiplicity | 4.8 | 5.2 | 5.6 | 2.4 | 2.4 |
| (2.0) | (3.1) | (3.6) | (2.2) | (2.3) | |
| Completeness (%) | 71.8 | 78.1 | 80.1 | 76.5 | 79.4 |
| (35.2) | (61.8) | (72.2) | (62.4) | (75.5) | |
| Mean (I/σ(I)) | 3.2 | 3.4 | 3.5 | 3.2 | 3.4 |
| (2.5) | (2.2) | (2.1) | (1.7) | (2.0) | |
| Rwork/Rfree | 36.1/39.2 | 37.1/38.4 | 34.6/37.3 | 27.3/31.9 | 26.2/30.8 |
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Values in parentheses reflect the highest resolution shell.
-
*
Data set used for final structure.
Table 3
Data collection and refinement statistics
| Data Collection | |
| Excitation voltage | 200 kV |
| Electron source | Field emission gun |
| Wavelength (Å) | 0.025 |
| Total dose per crystal (e−/Å2) | ∼6.8 |
| Frame rate (frame/s) | 1/6 |
| Rotation rate (°/s) | 0.09 |
| Angular range per frame (°/s) | 0.54 |
| No. crystals used | 1 |
| Total angular range collected (°) | ∼61 |
| Merging Statistics* | |
| Space group | P212121 |
| Unit cell dimensions | |
| a, b, c (Å) | 67.8, 172.1, 182.1 |
| α = β = γ (°) | 90 |
| Resolution (Å) | 21.2–3.2 (3.4–3.2) |
| Total reflections | 67,064 (9873) |
| Rmerge (%) | 17.5 (32.7) |
| Total unique reflections | 28,143 (4278) |
| Multiplicity | 2.4 (2.3) |
| Completeness (%) | 79.4 (75.5) |
| Mean (I/σ(I)) | 3.4 (2.0) |
| CC1/2 | 0.891 (0.555) |
| Data Refinement | |
| Reflections in working set | 26,732 |
| Reflections in test set | 1369 |
| Rwork/Rfree (%) | 26.2/30.8 |
| RMSD bonds (Å) | 0.006 |
| RMSD angles (°) | 1.05 |
| Ramachandran (%)† | |
| (Favored, allowed, outlier) | 96.6; 3.3; 0.1 |
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*
Values in parentheses reflect the highest resolution shell.
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†
Statistics given by MolProbity (Chen et al., 2010).
