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 0
  • 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, we need to tell Moorhen which map to use for the refinement

  • Maps → Click on the “○ Active” button of the masked map

The background of the button will turn black when it’s active.

Now define which atoms we want to refine (in this case, all of the nanobody)

  • 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

Moorhen Selection Panel

  • 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