Coot Cryo-EM Practical

Aim: we will fit a domain of the human respiratory complex I matrix arm starting from an empty
(part of the) map.

WARNING:: This is non-trivial, a familiarity with Coot is assumed. This is not like
the basic tutorial. This is approximately how I use coot. It is tricky because
fitting cryo-EM maps is tricky.

Put the scripting files in $HOME/.coot-preferences (make the directory if needed)

Using Coot:

• read in the map for EMD-6771

Let’s make a smoother map (EMD-6771 is not typical in that it is well sampled and
not obviously oversharpened). But let’s learn how to do it because that is often
not the case.

• Extensions → Modules s → Cryo-EM, Cryo-EM s → Multi-sharpen…

The runs refmac to make an MTZ file starting-map-xxx.mtz. Read it in.

• read 5xtb-sans-J.pdb

• Fetch the model for accession code 3wj7 from PDBe

• Use Draw → Sequence View and Edit → Copy Fragment to extract the protein
  A chain from this (only the protein part)

• Centre on the unmodelled density for the J chain

• Move the A fragment from 3wj7 to here

• Morph → Jiggle fit this chain (this can take ~60 seconds)

• Morph ----------- Morph by secondary structure here

  Does it fit? If not, try again. Maybe shift the centre of the molecule by a few Angstroms

• Using the ProSMART module, generate 4.3A self-restraints for this (now fitted) chain

• Chain-refine (Shift E)

• Calculate → Scripting → Python:
  set_alignment_gap_and_space_penalty(-6, -0.4)

• Calculate → Align and mutate the A-chain-fragment to the seqence of the J chain

• Use the Validation tools to find problematic regions of the model and fix them.

Hint 1: Density Fit analysis is a good tool to start off.

Hint 2: Use the sequence file J-chain.seq for alignment

End: You may notice that theres a ligand in the middle of the protein - it’s NDP.
Get an instance of NDP and fit it to the map using
Calculate → Other Modelling Tools → Ligand Fitting