8. CCP4 Coot Refinement protocol

Protocol designed to interactively fit and refine atomic structures, in real space, regarding electron density maps in Scipion by using Coot [Emsley et al., 2010]. This protocol integrates Coot 3D graphics display functionality in Scipion, supporting accession to Coot input and output data in the general model building workflow.

COOT, acronym of Crystallopgraphic Object-Oriented Toolkit, gathers several tools useful to perform mostly interactive modeling procedures and is integrated in CCP4 software suite (CCP4). Initially applicable to X-ray data, some modifications of Coot also allow to model atomic structures regarding electron density maps obtained from cryo-EM [Brown et al., 2015]. Additional instructions to use Coot can be found in COOT. Remark in COOT mouse and keyboard mouse requirements to get the best functioning.

• Requirements to run this protocol and visualize results:
  • Scipion plugin: scipion-em
  • Scipion plugin: scipion-em-ccp4
  • CCP4 software suite (from version 7.0.056 to 7.1)
  • Scipion plugin: scipion-em-chimera
• Scipion menu:

  Model building -> Flexible fitting (Fig. 8.2 (A))
  

  Fig. 8.2 Protocol ccp4-coot refinement. A: Protocol location in Scipion menu. B: Protocol form.

• Protocol form parameters (Fig. 8.2 (B)):
  • Input section
    • Input Volume/s: At least one or several electron density maps previously downloaded or generated in Scipion. The density volume regarding to which an atomic structure has to be modeled has to be included in this volume list.
    • Normalize: Parameter set to “Yes” by default to perform normalization of map electron density levels according to Coot requirements ([0, 1]). This normalization approximates cryo-EM density data to maps obtained from X-ray crystallography because it diminishes Z-score (number of standard deviations) variation of map values.
    • Atomic structure to be refined: Mandatory param to load an atomic structure previously downloaded or generated in Scipion. This structure will be fitted and refined according to a particular density volume.
    • Other reference atomic structures: Additional atomic structures previously downloaded or generated in Scipion that may be helpful in the refinement process.
  • Help section
    This section contains Coot commands to make easier some interactive refinement steps and to save refined atomic structures. Their reference volumes will be saved by default with the refined atomic structures. Here you are an overview of these commands:
    • Automatically moving from one chain to another in an atomic structure:
      • Press x in the keyboard to move from one chain to the previous one.
      • Press X to change from one chain to the next one.
    • Initializing global variables:

      Press U in your keyboard.
    • Semi-automatic refinement of small groups of residues (10 to 15):
      As soon as Coot protocol is executed, the text file coot.ini will be saved in the project folder /Runs/00XXXX_CootRefine/extra/ (Fig. 8.7 (1, 2)). This file content has to be modified according to our atomic structure model in this way:
      • imol: #0 has to be replaced by the number of the molecule that has to be refined. This number appears detailed in Coot main menu Display Manager (Fig. 8.3 (B, red arrow)).
      • aa_main_chain: A has to be replaced by the name of the molecule chain that has to be refined.
      • aa_auxiliary_chain: AA, name of the small chain of 10-15 residues, can be optionally replaced by other name.
      • aaNumber: #100 has to be replaced by the position of the residue from which the refinement has to start.
      • step: #10 will be replaced by the desired small step of residues that gets flexible enough to select other conformation of this auxiliary chain.
      Save coot.ini text file after modifying it. Go to the residue position indicated in aaNumber, initialize global variables with U, and pres z or Z in the keyboard to refine those aaNumber residues upstream or downstream, respectively.
    • Printing Coot environment:

      Press E in the keyboard.
    • Saving an atomic structure after an interactive working session with Coot:
      Coot Python Scripting window will be opened with Coot main menu Calculate -> Scripting… -> Python… (Fig. 8.3 (A)). By writing scipion_write(), molecule #0 will be saved by default in Scipion. Molecule number can be checked in Coot main menu Display Manager (Fig. 8.3 (B, red arrow)). Saving the molecule this way is equivalent to press w in the keyboard.
      The number #n of the specific molecule has to be written in brackets to save any other molecule than #0.
      Although the name of the saved atomic structure is coot_XXXXXX_Imol_YYYY_version_ZZZZ.pdb by default (XXXXXX is the protocol box number, YYYY the model number and ZZZZ the number of times that the molecule has been saved), other names/labels of your preference are also allowed. That name/label has to be introduced with scipion_write() command, as it is detailed in the example (Fig. 8.3 (A)). The addition of .pdb extension is not required.
      If no more interactive sessions with Coot are planned, after saving the atomic structure, Coot can be definitively closed by pressing e in the keyboard.
      

      Fig. 8.3 Protocol ccp4-coot refinement. A: Saving labeled atomic structure with Coot Python Scripting window. B: Display Manager window.

• Protocol execution:
  Adding specific map/structure label is recommended in Run name section, at the form top. To add the label, open the protocol form, press the pencil symbol at the right side of Run name box, complete the label in the new opened window, press OK and, finally, close the protocol. This label will be shown in the output summary content (see below). If you want to run again this protocol, do not forget to set to Restart the Run mode. However, if you want to restart the protocol in the last point that you let it before and continue working with the last file saved in Coot, set to Continue the Run mode.
  Press the Execute red button at the form bottom.
  Coot graphics window will be opened after executing the protocol. Electron density maps and atomic structures are shown. Although steps to follow depend on the specific operation to carry out, a list of basic initial tasks and tools could be helpful:
  • Check maps and atomic structures definitively loaded in Coot:

    By opening Display Manager window (Coot main menu) (Fig. 8.3 (B)).
  • Set parameters appropriate to visualize them:
    Electron density maps are sometimes more difficult to visualize. Moving mouse scroll-wheel forward and backward increases or reduces, respectively, map contour level. If the volume is still invisible, check if map and atomic structures are properly fitted. The radius of the density sphere can be modified in Coot main menu Edit -> Map Parameters … -> Global map properties window.
  • Check chain names of each atomic structure, and edit them if needed in Coot main menu Edit -> Change Chain IDs….
  • Set the text file coot.ini (Fig. 8.7 (2)), edit it and save it if needed.
  • Set refinement conditions:

    Click Refine/Regularize control button (upper right side of Coot graphics window) (Fig. 8.4 (1)) and select the four restriction types in Refinement and regularization Parameters window (2).
    

    Fig. 8.4 Protocol ccp4-coot refinement. Refinement and regularization Parameters window.

  Once those basic parameters are set up, some steps to follow in the refinement process are:
  • Check validation parameter windows to have an idea of controversial areas and quality of the fitting:

    Go to Coot main menu Validation -> Ramachandran Plot, Validation -> Density fit analysis and Validate -> Rotamer analysis. Validation windows have to be checked throughout the refinement process.
  • Refine the ends of each chain. Basic interactive refinement process requires several steps:
    • First, go to an atom included in the area that is going to be refined:

      Go to Coot main menu Draw -> Go To Atom… and select chain and atom.
    • Assess electron density in that area, and consider the possibility of processing part of the residues.
    • Click the button Real Space Refine Zone (upper right side of Coot graphics window) (Fig. 8.5 (A) (1)) to put it active. Next, click two residues of the chain (2 and 3). A second flexible grey chain overlaps the starting chain. That grey chain can be moved in order to get a different conformation according to the density map (hidden in Fig. 8.5 (A)).
    • If refinement parameters get acceptable values, press Accept in Accept Refinement? window (Fig. 8.5 (B)).
      

      Fig. 8.5 Protocol ccp4-coot refinement. (A) Interactive refinement of the chain fragment between residues 2 and 9. (B) Accepting refinement window.

  • Refine each chain following instructions from Help section:
    • Go to the residue aaNumber (main menu Draw -> Go To Atom…).
    • Initialize global variables.
    • Repeat this loop until reaching the end of the chain:

      1.- Press z in the keyboard.

      2.- Inspect one by one, and fit to the volume density, every residue from the small auxiliary chain.

      3.- Accept the refinement.
    • Check validation parameters to focus refinement in specific chain areas (Coot main menu Validate -> Density fit analysis).
  • After finishing refinement of every chain, save the structure (press e if Coot has to be definitively closed and not interactive anymore).
  • Close Coot graphics window.
• Visualization of protocol results:
  After executing the protocol, press Analyze Results and ChimeraX graphics window will be opened by default. Atomic structures and volumes are referred to the origin of coordinates in ChimeraX. To show the relative position of atomic structures and electron density volumes, the three coordinate axes are represented; X axis (red), Y axis (yellow), and Z axis (blue) (Fig. 5.3). Coordinate axes, volume, and first atomic structure are model numbers #1, #2, #3, respectively, in ChimeraX Model Panel. Every atomic structure saved during refinement process will appear in Model Panel (Fig. 8.6). If you want to visualize results in Coot graphics window you only have to open the protocol in the last point that you let it before and set to Continue the Run mode. Close the Coot protocol without saving anything in this case.
  

  Fig. 8.6 Protocol ccp4-coot refinement. Coot results visualized in ChimeraX.

  Since Scipion projects keep every intermediate atomic structure partially refined (Fig. 8.7 (1, 3), users can include any of them in successive following modeling workflow steps performed in Scipion (Fig. 8.8).

  

  Fig. 8.7 Protocol ccp4-coot refinement. Browse content after several runs of interactive Coot protocol.

  

  Fig. 8.8 Protocol ccp4-coot refinement. Scipion window that allows to select any of Coot partially refined structures.

• Summary content:
  • Protocol output (below Scipion framework):
    • Each Coot intermediate atomic structure partially refined (#n):

      ccp4 - coot refinement -> label name selected by the user or common output name (coot_XXXXXX_Imol_YYYY_version_ZZZZ.pdb);

      AtomStruct (pseudoatoms=False, volume=False).

      Pseudoatoms is set to False because the structure is made of atoms instead of pseudoatoms. Volume is set to False because no electron density map is associated to the atomic structure.
    • Each Coot input map (saved by default):

      ccp4 - coot refinement -> output3DMap_XXXX;

      Volume (x, y, and z dimensions, sampling rate).
    • SUMMARY box for each Coot intermediate atomic structure partially refined:

      label name selected by the user or common output Coot name (coot_XXXXXX_Imol_YYYY_version_ZZZZ.pdb)

      Idem for maps.