.. _buildingunitcell:

Building the asymmetric unit
============================

| Once we have selected the *models* for *metHgb* :math:`\alpha`
  and :math:`\beta` subunits (see the workflow branches to have
  :math:`\alpha` and :math:`\beta` subunits; :numref:`model_building_scipion_workflow_whole_reconstruction`), we can regenerate the
  smallest asymmetrical element of the starting map. With this aim we
  are going to use protocols to operate with atomic structures (**chimerax-operate** or **atomstructutils-operator**), to refine them both manually (**ccp4-coot refinement**) and automatically (**phenix-real space refine**) and to validate them (**phenix-validation_cryoem** and **phenix-emringer**). A brief schema of the main steps of this part of the workflow can be seen in :numref:`model_building_scipion_workflow_whole_reconstruction`. Take into account that in real live probably
  many more steps of refinement and validation will be required.

.. figure:: Images/Fig73.svg
   :alt: *Scipion* framework detailing the workflow to reconstruct the structure of the map asymmetric unit (blue arrow) and the whole atomic structure (red arrow).
   :name: model_building_scipion_workflow_whole_reconstruction
   :align: center
   :width: 100.0%

   *Scipion* framework detailing the workflow to reconstruct the structure of the map asymmetric unit (blue arrow) and the whole atomic structure (red arrow).

-  | Protocol to join the *metHgb* :math:`\alpha` and :math:`\beta`
     subunits in a unique atomic structure:

   | Two protocols can be used for this purpose (**chimerax-operate** or **atomstructutils-operator**) and the result should be identical.
   | Before starting, nevertheless, be sure that you have two atomic
     structures and each one includes an only chain with a different
     *id*. Remember that chain *ids* may be changed for other chain
     *ids* in *ChimeraX* and *Coot*.
   | Secondly, it could be very convenient to change the *Scipion* output label of
     each subunit, in order to follow them easily in *Scipion*. According to :numref:`model_building_scipion_workflow_edition`
     go to the Summary of the two final protocols that allow to generate
     those atomic structures and press the black arrow (C) to select the
     option *Edit*. Type the new output name of the structures
     (*HBA_refined* (D) and *HBB_refined* (E), respectively).

   .. figure:: Images/Fig75.svg
      :alt: A. Zoom in on :numref:`model_building_scipion_workflow_whole_reconstruction`. B. Summary of the protocol box from *PHENIX real space refine* (:math:`\alpha` in (A)). Red arrow points at the *Scipion* output name. C. Menu opened pressing the output black arrow of the Summary. D. New name of the *Scipion* output in the Summary. E. Summary from the protocol box *PHENIX real space refine* (:math:`\beta`) after applying the same edition process.
      :name: model_building_scipion_workflow_edition
      :align: center
      :width: 100.0%

      *A*. Zoom in on :numref:`model_building_scipion_workflow_whole_reconstruction`. B. Summary of the protocol box from *PHENIX real space refine* (:math:`\alpha` in (A)). Red arrow points at the *Scipion* output name. C. Menu opened pressing the output black arrow of the Summary. D. New name of the *Scipion* output in the Summary. E. Summary from the protocol box *PHENIX real space refine* (:math:`\beta`) after applying the same edition process.

   | Then, open again *ChimeraX operate* protocol and following
     the already indicated instructions, include the *models* of
     *metHgb* :math:`\alpha` and :math:`\beta` subunits in params
     *Atomic structure* and *Other atomic structures*, respectively (:numref:`model_building_chimerax_asymm_unit_1` (A)). Firstly, check that both *models* are perfectly fitted in the map asymmetric unit. Otherwise, apply the command *fit inMap*, as it was previously shown. Next, create a single atomic structure by joining models *#3* and *#4* in *ChimeraX Models* panel. To
     generate a combined *model* write in the command line:

     ::

         scipioncombine #3,4

   | The new model *#5* is shown in *ChimeraX Models* panel (:numref:`model_building_chimerax_asymm_unit_1`).
     Finally, save this fitted structure writing in *ChimeraX* command line:

     ::

         scipionwrite #5 prefix asymmetric_unit_model_

   .. figure:: Images/Fig76.svg
      :alt: A. Completing the protocol **chimerax-operate** with the atomic structures *HBA_refined* and *HBB_refined*. B. *ChimeraX* graphics window showing the combined *model #5*.
      :name: model_building_chimerax_asymm_unit_1
      :align: center
      :width: 100.0%

      *A*. Completing the protocol **chimerax-operate** with the atomic structures *HBA_refined* and *HBB_refined*. B. *ChimeraX* graphics window showing the combined *model #5*.

-  | Protocols to refine the new combined structure generated:

   | At this point refinements could cover specially the overlapping
     area between the two chains. Help yourself with the *Coot* tools of
     *Validate* in the main menu, as well as the visualization tools of
     *PHENIX real space refine* protocol.

-  | Validation protocols to select the best *model* of the human
     *metHgb* unit cell:

   | Validate the new combined structure generated is recommendable
     before continuing with the next steps in the
     workflow. *EMRinger* and *Validation CryoEM (MolProbity)* validation statistics should
     be computed for the new *model* of human *metHgb* asymmetric
     unit, generated by combining *metHgb* :math:`\alpha` and :math:`\beta` subunits. Appendix :ref:`Solutions <app:solutions>` (:ref:`Question11 <question11>`) contains a statistics table for the unit cell *model* (Table 8). We can try to improve those statistics by additional refinement processes. By
     performing refinement in real space with *PHENIX* some of the
     statistics could result improved. Table 8 contains also RMSD values
     computed in a similar way as we have seen for :math:`\alpha` and :math:`\beta` subunits, considering as fixed structure chains A and
     B from *5NI1* atomic structure. To continue with the modeling
     process we can select the unit cell *model* generated by *PHENIX real space refine* because most of its validation statistics show the best values (*CC(mask)*, *EMRinger score* and *MolProbity* values). Exceptionally, RMSD regarding the published structure yields the worst value.