Coot Cryo-EM Tutorial: Part 2: Molecular Extraction

Part 2 of this tutorial is similar to Part 1, but the setup is a little different.

1: Find the Nanobody

Now we want to model the nanobody domain.

We have done a BLAST search for the seqence of our nanobody and know that the structure was published already in a different complex - it’s the “E” chain in the entry 6GDG. So let’s fetch that structure, extract the “E” chain and then fit it to our reconstruction.

  • First centre the view on the centre of the nanobody/immunoglobulin domain. You should be able to do this by eyeballing it and using middle-mouse click and drag - but if not, then:
  • Calculate → Scripting → Python
  • set_rotation_centre(121, 71, 102)
  • Press “Enter” to execute.

2: Fetch the Reference Structure

Now let’s download the reference structure:

  • File → Fetch PDB using Accession Code → 6gdg → Get it
  • {Coot downloads 6gdg and moves the centre of the screen it its centre}
  • Draw → Undo Last Navigation
  • {Coot changes the centre of the screen to were we had been before loading 6dgd}
  • Edit → Copy Fragment
  • Change the molecule selector combobox to the most recently loaded molecule: i.e. coot-download/6gdg.cif
  • Atom Selection for fragment → //E
  • Check the Move new molecule here checkbutton
  • OK
  • {Coot places the “E” chain at the centre of the screen}

We no longer need to see 6dgd.cif so use the Display Manager to undisplay it.

3: Jiggle-Fit

Let’s make the “E” chain a bit more clear by changing the molecular representation:

  • Ctrl Shift mouse-forward-scroll
  • {Coot changes to CA-bonds mode}
  • Cryo-EM → Jiggle-Fit with Fourier Filter
  • {Coot changes the “E” chain to the correct orientation to fit the map}

4: RSR Morph Refinement

Now we proceed in a similar way to Part 1:

  • Restraints → Generate All-Molecule Self Restraints 5.0
  • Undisplay Extra Restraints
  • R/RC → Refinement Weight → Estimate
  • More Control Geman-McClure weight to 0.01 → OK
  • Refine → Chain Refine
  • {watch and wait}
  • Accept

Now let’s reduce the weight on the GM restraints:

  • More Control Geman-McClure weight to 0.1 → OK

This should allow the model to make local/smaller scale adjustments to better fit the map

  • Refine → Chain Refine
  • {watch and wait}
  • Accept

5: Merge

Merge this into the working.pdb molecule:

  • Edit → Merge Molecules
  • Check the button for Atom selection from 6dgd.cif
  • Change the molecule selector combobox to working.pdb
  • Merge