7. 3D Map preprocessing
The Fig. 7.1 shows the workflow that we are going to detail in this section.
7.1. Map sharpening
7.1.1. Sharpening with LocalDeblur
ShowJ, the default viewer, allows visualize the mask with shape similar to the starting volume (Fig. 7.3).
Note
In case you would like to use a previous computed mask, you can do it simply by importing it using the protocol import mask (Appendix Import mask).
Execute this protocol (Fig. 7.4 (5)) and analyze the results (6). The menu of results (Fig. 7.5 (A)), among other views, shows the histogram of local resolutions (1) and the resolution map in (2). The histogram of resolutions, which displays the number of map voxels showing a certain resolution, allows to conclude that the majority of voxels evidence a resolution between 3.2 and 3.5 Å, quite close to the published map resolution (3.2 Å). The resolution map shown by ChimeraX details the resolution of each voxel (Fig. 7.6). The bar on the left indicates the color code for resolution values.
Local resolution values of the input map allow to compute the sharpened map by the xmipp3-localdeblur sharpening protocol, which implements an iterative step descending method that doesn’t require initial model. To run this method, open the protocol (Fig. 7.7 (1)) and include the starting map (2) and the map of resolution values (3), maintaining the default values for the rest of parameters (4, 5).
After two iterations, the sharpening algorithm reached the convergence criterion, i.e. a difference between two successive iterations lower than 1%, and stopped. The two maps obtained in the respective iterations can be observed with ShowJ by clicking the black arrow shown in Fig. 7.7 (7) with the right mouse botton and selecting Open with DataViewer. Resulting map for each iteration will be shown, as indicated in Fig. 7.8. Visualization in ChimeraX is also possible selecting Open in the menu option File (Fig. 7.8 (1)).
Additionally, by clicking Analyze Results (Fig. 7.7 (6)) the sharpened map obtained after the second iteration, i.e. the last map, can be also visualized and compared with the initial one in ChimeraX (Fig. 7.9).
7.1.2. Sharpening with DeepEMhancer
DeepEMhancer is an alternative automatic sharpening method based on deep learning [Sanchez-Garcia et al., 2020], implemented in Scipion in the protocol xmipp3-deepEMhancer (Appendix DeepEMhancer Sharpening). Open this protocol (Fig. 7.10 (1)) and complete it as indicated. Since only the refined map is available, we are not going to use half maps (2). Include your map (3), the type of normalization desired (4) and the deep learning mode to use (5), in this particular case highRes due to the map high resolution.
After executing the protocol (Fig. 7.10 (6)), we can check the results (7). ChimeraX viewer will open and show the sharpened map compared with the initial one (Fig. 7.11).
7.2. Comparison of maps
Realize that at this point we have generated two optimized maps derived from the initial one. Additionally, some other maps could have been obtained using other map optimization methods. A comparison among them would be interesting to consider which one(s) of them should be used as input in next steps of modeling workflow. The ideal map for tracing the atomic structure should include as many details and connections as possible and, at the same time, preserve the density areas of the initial map. In other words, we can use the best sharpened map (with higher resolution) corroborating that it does not make up new densities, absent in the starting map. Nevertheless, selecting “the best” sharpened map could be difficult sometimes, especially if the map is very big or there are some regions optimized in one of the sharpened maps and other areas optimized in the other one. In that case, you can use several maps at the same time, having all of them perfectly aligned according to the same origin of coordinates.
In the tiny example shown in this tutorial we are working with a high resolution map and there are almost no differences in resolution between the starting map and the two derived sharpened maps, although this is not usually the case in real life. In this quite uncommon case the initial unsharpened map would be enough to trace the atomic structure. However, in order to detail the method, the starting map and their two sharpened ones will be used simultaneously.
7.3. Extraction of the asymmetric unit map
After executing the protocol (Fig. 7.12 (9)), the resulting expanded ASU can be observed (10) with ChimeraX (Fig. 7.13). Note the additional expanded volume of the ASU on the left side of the figure. The ASU itself, on the right side, constitutes the half volume. Since the total volume contains the structure of four proteins, we can anticipate that this smaller asymmetrical subunit of the initial volume contains two proteins, one \alpha and one \beta subunit. Then, the respective structures of these two proteins could be fitted in the map ASU simultaneously or in successive modeling workflow steps.