Getting Started: Load the Map and Model

Get the reference files:

- https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/files/32143-partial-model.pdb
- https://ftp.ebi.ac.uk/pub/databases/emdb/structures/EMD-32143/map/emd_32143.map.gz

At the moment, you will need to gunzip the map.

  • File → Coordinates → 32143-partial-model.pdb → Open
  • File → CCP4/MRC Map → Choose File emd_32143.map → Open
  • Maps → Pull across the Radius to 77

  • (you can also use ‘[’ and ‘]’ to increase and decrease the map radius)

  • Map Tools → Sharpen/Blur map
  • B-factor to apply should be zero
  • Turn on use resample and use the resample factor 1.4 → OK
  • Maps → Click on the “Gear” icon on the (newly-created) Masked map → Draw settings → Activate “Lit lines”

You will see that this new map is more easy to interpret.

  • Delete the first map:
  • Maps Click on the “Gear icon” of the panel for the first map and Delete Map.

Rigid-Body Fitting

  • Undisplay the new map:
  • Click on the “Eye” icon

We are, at the moment, only interested in the parts of the map for which we don’t currently have a model. So now mask the map using the (partial) model (the parts of the model that we have already fitted):

  • Map Tools → Map masking… → OK

You should now see a map with three unfitted parts

  • the hormone
  • a WD40 domain
  • a nanobody domain

Moorhen 3 Domains

Fitting the Nanobody

We have done a BLAST search for the sequence 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.

Fetch the Reference Structure:

  • File → Fetch from Online Services → 6gdg

Now make the fragment we need from that:

  • Edit → Copy Fragment
  • Change “From molecule” to 6gdg
  • Selection to Copy: //E
  • OK

So now we have the “E” chain floating around in space…

Delete the 6gdg model - we don’t need it any more:

  • Models Click on the “Gear” icon of the 6gdg molecule and Delete molecule

Change the representation of the ‘//E’ chain

  • Models → Unclick the “Bonds” button and click the “Ribbons” button

Jiggle Fitting

Centre the view on the nanobody domain by “eyeballing” it and using middle-mouse click and drag. Rotate the view to check that you have found the approximate middle from various directions.

  • Edit → Move Molecule Here →
  • Select the molecule 6gdg fragment → OK

Now to do the actual fitting:

  • Calculate → Jiggle Fit with Fourier Filtering
  • Choose molecule 6gdg fragment
  • Change the map to Map 1 masked
  • Add an “Atom selection” as //E
  • Change the number of trials to 310

Moorhen will think for a few seconds and then fit the nanobody into the density.

Refinement

  • Change the ribbon representation back to bonds:
  • Models → Unclick the “Ribbons” button and click the “Bonds” button

Now set-up the local distance restraints:

  • Calculate → Generate Self Restraints
  • Selection is “Molecule”
  • Molecule is 6gdg fragment
  • Max Dist change to 5.1

Moorhen generates extra restraints (shown in grey lines)

  • Maps → “Gear” icon of the masked map → set map weight
  • 1830 (it should be the default) → Set

To refine this domain/chain:

  • Edit → Create a selection
  • Molecule is 6gdg fragment
  • Atom Selection is // (i.e. “all atoms”)
  • OK

You will see that the nanobody is now highlighted with green bonds and atoms. A dialog will appear at the middle top

  • Click on the “Refine” icon (top left)
  • Moorhen refines the nanobody
  • Accept the modification

Merge

After refinement we should merge the nanobody into the main molecule.

  • Edit → Merge Molecues
  • “From molecule” is 6gdg fragment
  • “Into molecule” 32143 partial model
  • OK

Now save the combined model:

  • Models → Click on the “Download” icon of the 32143 partial model