15. Summary of results and submission

Once we have selected the best model of the whole human Hgb and obtained good validation scores from EMRinger, MolProbity, and other validation programs, and we have checked that we have the whole volume density modeled, we are ready to submit the electron density map and its atomic interpretation to public databases and to make public our results.

15.1. Submission to public databases

Although submission of cryoEM maps and derived atomic structures to databases has to be done by direct online request (wwPDB OneDep System), Scipion may contribute to organize the submission records. The protocol export to DB allows to perform this task (Appendix Submission to wwPDB). By using this protocol we can save the files that you have/want to submit to databases in a labelled folder and in the appropriate format for submission. Fig. 15.1 details the protocols of the modeling workflow involved in this task.

*Scipion* framework detailing the workflow to submit *cryo-EM* results to databases.

Fig. 15.1 Scipion framework detailing the workflow to submit cryo-EM results to databases.

When you submit the map and the model of a cryo-EM experiment, besides these two records, an image of the map is also mandatory to submit. Other maps, such as half maps or postprocessing-sharpening maps, as well as maks, are also recommended to submit. In addition, the FSC file is strongly encouraged. As you can see in Fig. 15.1, we can provide directly from the workflow the map and the model, as well as the two sharpening maps. The map image can be attached from a file. We lack, however, from the FSC file, since the FSC file is usually generated during the map reconstruction process starting from the half maps, for example with the xmipp3-resolution 3D protocol (Fig. 15.1, red arrow). To compute the FSC file we could download the half maps from the database (PDBe EMD-3488) selecting the zip Bundle (Fig. 15.2 (red arrow)).

*EMDB* entry *3488* in *PDBe*

Fig. 15.2 EMDB entry 3488 in PDBe

The zip folder contains the FSC file (emd_3488_fsc.xml) and the map image (emd_3488.png) but, unfortunately, lacks of half maps. Then, you can use any two half maps and compute the FSC file, just to submit it with the rest of the files.

To save all the relevant files in a single labelled folder, open the export to DB protocol (Fig. 15.3 (1)), and complete the form with the Scipion elements to export: Main map (2), Additional maps: “Yes” (3), the two sharpened maps as additional maps (4), the FSC file if you count on it (5), Atomic structure (6) and Image (7), previously saved in a known folder. Then, write the name of the exportation directory path, or find it with the browser on the right. All submission files will be saved in the directory selected (8). A directory name related with the submission (number, date, project,…) is recommended.

Saving files for submission to EMDB with protocol **export to EMDB**

Fig. 15.3 Saving files for submission to EMDB with protocol export to EMDB

After executing the protocol (9), you can check that all files are saved in the given directory. No additional visualization tools have been included in this protocol.

15.2. Publication of results

Since the atomic interpretation of a certain macromolecule will be probably the starting point of relevant mechanistic or biomedical studies, summaring and organizing our results constitutes the first step to draw the conclusions that will be made public by journals and talks. Many different questions can be posed based on the atomic structure. Here we are wondering about interactions among members of the macromolecule. To answer this question we have included in Scipion the protocol chimerax-contacts to identify the residues involved in contacts between any couple of interacting molecules. “contacts” involve atoms within favorable interaction distances. Unfavourable contacts or severe clashes, in which atoms are too close together, although discarded by default in the final list of ‘contacts’’, may also be shown by using appropriate advanced parameters, as you can see in Appendix CHIMERAX Contacts.
As an example, in this tutorial we are going to learn how to get atom contacts of human haemoglobin metHgb atomic structure 5NI1, associated to the starting map EMD-3488. This structure was already downloaded from PDB by using the protocol import atomic structure (Fig. 15.4 (1)). According to the aim of the analysis, two possible scenarios and the respective workflows can be considered to compute contacts: a) infering all contacts between any couple of members of the whole macromolecule (Fig. 15.4 (3)); b) infering all contacts between any couple of members of the asymmetric unit, and between one member of the asymmetric unit and another component from a neighbor asymmetric unit (Fig. 15.4 (5)).
*Scipion* workflows inside the red box to get contacts between any two chains of a macromolecule (3) and between any two chains of the asymmetric unit, and between any chain of the asymmetric unit and a chain of a neighbor asymmetric unit (5).

Fig. 15.4 Scipion workflows inside the red box to get contacts between any two chains of a macromolecule (3) and between any two chains of the asymmetric unit, and between any chain of the asymmetric unit and a chain of a neighbor asymmetric unit (5).

Since the penultimate step of the second workflow (Fig. 15.4 (4)) requires applying symmetry, we are going to start moving the structure to match its symmetry center to the origin of coordinates using the protocol phenix-dock in map as we did previously (Fig. 10.2), including the whole starting map of the human metHgb and the imported atomic structure 5NI1 as Input map and Input atom structure, respectively.
Secondly, we are going to extract the structure of the asymmetric unit of the docked 5NI1 structure using the protocol chimerax-operator as it is indicated in Fig. 15.4 (4). Complete the protocol form including the last docked structure 5NI1 as Atomic structure. After executing the protocol, the graphics window will open. You can select and save the atomic structure of the map asymmetric unit writing in the command line:
select #2/A,B
save /tmp/chainAB.cif format mmcif models #2 selectedOnly true
open /tmp/chainAB.cif
scipionwrite #3 chainAB_
exit
  • CASE A: Contacts between any couple of members of the whole macromolecule (Fig. 15.4 (3)):
    This option allows to get all contacts between all couples of members of the macromolecule. In the case of the human metHgb we have depicted all those possible contacts in Fig. 15.5 (A).
    Schema of the human haemoglobin *metHgb* showing protein contacts between couples of chains of the whole macromolecule (A) and contacts obtained by applying symmetry to the asymmetric unit (B).

    Fig. 15.5 Schema of the human haemoglobin metHgb showing protein contacts between couples of chains of the whole macromolecule (A) and contacts obtained by applying symmetry to the asymmetric unit (B).

    The protocol chimerax-contacts can be used to obtain the contacts depicted. Open this protocol (Fig. 15.6 (1)) and fill in the first Input (2) in which no symmetry will be applied. Include the docked 5NI1 structure (4) as Atomic structure. Use the wizard on the right to label the molecule chains (5) as they appear in the adjacent window, and execute the protocol.

    Filling in the protocol **chimerax-contacts** form with two different inputs: (2) to get atom contacts between couples of chains within the whole *metHgb*; (3) to get contacts between any couple of chains within the asymmetric unit, and “non-redundant“ contacts between the asymmetric unit and another chain of a neighbor asymmetric unit of the human haemoglobin *metHgb*.

    Fig. 15.6 Filling in the protocol chimerax-contacts form with two different inputs: (2) to get atom contacts between couples of chains within the whole metHgb; (3) to get contacts between any couple of chains within the asymmetric unit, and “non-redundant“ contacts between the asymmetric unit and another chain of a neighbor asymmetric unit of the human haemoglobin metHgb.

    After executing the protocol, all atom contacts between the couples of proteins indicated in Fig. 15.5 (A) can be visualized by clicking Analyze Results (Fig. 15.7 (A)).

    (A) Display of results of atom contacts between couples of chains within the whole *metHgb*; (B) Display of results of atom contacts between couples of chains within the asymmetric unit, and ”non-redundant“ contacts between a chain of the asymmetric unit and another chain from a neighbor asymmetric unit of the human haemoglobin *metHgb*.

    Fig. 15.7 (A) Display of results of atom contacts between couples of chains within the whole metHgb; (B) Display of results of atom contacts between couples of chains within the asymmetric unit, and ”non-redundant“ contacts between a chain of the asymmetric unit and another chain from a neighbor asymmetric unit of the human haemoglobin metHgb.

    The viewer window of the protocol ChimeraX contacts display different results (Fig. 15.7 (A)):

    • 3D Visualization box: Final atomic structure considered to compute contacts that can be visualized with ChimeraX. Press the eye (1) to open the structure shown on the right.
    • Interacting chains box: Summary list of all interacting chains, similar to the list shown on the right of the Fig. 15.5 (A). Press the eye to open it (2).
    • Contacts between interacting chains box: In addition to the possibility of changing the order of the interacting chains in the display, as well as the maximal distance between residues to group them, this box allows to select couples of interacting chains (4) and inspect in detail the contacts between them pressing the eye on the right (3).
  • CASE B: Contacts between any couple of members of the asymmetric unit and ”non-redundant“ contacts between one member of the asymmetric unit and another one from the neighbor asymmetric unit (Fig. 15.4 (5)). This second asymmetric unit has been obtained by applying symmetry with the protocol chimerax-contacts. Then, “non-redundant” interaction means any interaction that can not be inferred by symmetry. The Fig. 15.5 (B) shows the total number of interactions of our example. The interactions between the chain B of the asymmetric unit (model #1.1) and the chain A of the neighbor asymmetric unit (model #1.2) are symmetric to the interactions between chain A of the asymmetric unit (model #1.1) and chain B of the neighbor asymmetric unit (model #1.2). Since those interactions can thus be inferred by symmetry, they are “redundant” and are absent of the final list of contacts.
    Similarly to the case A, the protocol form has to be open (Fig. 15.6 (1) ) and completed as indicated in the second Input (3). Include the asymmetric unit structure saved with the protocol ChimeraX operate (6), use the wizard on the right (7) to label the chains as it is shown on the right and, finally, include the respective type of symmetry of the human metHgb (8).
    Like in the case A, after executing the protocol all non-redundant atom contacts between any couple of proteins indicated in Fig. 15.5 (B) can be visualized by clicking Analyze Results (Fig. 15.7 (B)). Besides the lower number of contacts displayed, remark that a relevant difference between the results of the case A and the case B is the final atomic structure visualized with ChimeraX, which discriminates between the starting asymmetric unit and the second one generated by symmetry.
    NOTE: This second possibility of getting protein contacts observed in the case B is extremely useful when you have a big asymmetric unit, for example of a virus, and you are interested in contacts among proteins within the asymmetric unit and with other adjacent asymmetric units.