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 a web browser, download the following

Using Coot:

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.

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

If you can’t make it Jiggle correctly, then the “real” centre is at (193,208,286). Move the fragment so the middle of the molecule is there and Jiggle fit will work.

The real sequence is quite different to this model, let’s strip off the side chains

We still have the wrong sequence, let’s apply the new one:

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