Coot Cryo-EM Tutorial: Part 3: De Novo Model-Building

Now it’s time to build the parathyroid hormone (or at least, the part of it that we can see in the reconstruction)

1: Build a Poly-Ala Helix

  • Centre on the middle of the helix that is the part of the map for the parathyroid hormone. You should be able to eyeball this, but if not:
  • Calculate → Scripting → Python
  • set_rotation_centre(121, 71, 102)
  • Press “Enter” to execute.
  • Calculate → Other Modelling Tools → Place Helix Here
  • {Coot creates a helix}
  • Refine → Chain Refine
  • Accept

2: Add Sidechains

Now let’s put on some sidechains:

  • Calculate → Assign Sequence… → 1: Associate Sequence File…
  • Change the molecule selector combobox to the most recently created molecule: i.e. Helix-5 (your molecule number may be different)
  • Use the File… button to find and open the file parathyroid-hormone.pir
  • OK
  • Cryo-EM → Assign Sequence Based on Associated Sequence
  • {Coot adds sidechains}
  • Refine → Chain Refine
  • For me, the side chain for LYS 58 is in the wrong direction, so pull it into the correct tube (the density is not great)
  • Bring TRP 54 to the centre of the screen and
  • Backrub Rotamer Fit
  • {Coot flips the sidechain into its density}
  • Accept

3: Merge

Merge this into the working.pdb molecule:

  • Edit → Merge Molecules
  • Check the button for Helix-5 (or whatever your molecule number was)
  • Change the molecule selector combobox to working.pdb
  • Merge

4: How did we do?

Now we can compare your model with the published model, 7vvl.

  • Download the model using File → Fetch Model using Accession Code→ 7vvl → OK
  • Compare and contrast.