By Ana Casañal & Paul Emsley

This tutorial is designed for 0.9.1 or later

1: Start

  • Open a Terminal
  • cd s2c2_ccpem_workshop
  • cd coot_tutorial_1
  • if you have an NVIDIA graphics card:
  • export __GL_FSAA_MODE=5
  • Start Coot:
  • coot
  • Keep the terminal window where Coot is running always visible. You will see that as many of the algorithms run they will be writing useful information.

1.1 Load the Tutorial Data

  • Calculate → Load Tutorial Model and Data
  • Open the Display Manager
  • Delete Map for the difference map (the one with labels “DELFWT PHDELWT”)

1.2 Representation

  • Edit → Map Parameters…
  • A Map Radius of 70Å is a lot of map, useful for a cryo-EM reconstruction, but for now we are focussing on details:
  • Map Radius x-ray → 16 {sensible for crystallographic map}
  • Edit → Map Colour…
  • Change the map colour {see that dark pastel colours are the best}
  • Display Manager → Properties → Solid/Transparent
  • Tweak the Opacity %age
  • Useful? Maybe. (About 30% seems best)
  • Back to Standard Lines

Note: if you have a speedy computer, then you can make a copy of the map (Calculate → Map Tools → Copy Map Molecule…) and use Standard Lines for one and Solid/Transparent for the copied map - that looks quite good and can be helpful. It can be laggy though with a big radius, and that is the opposite of useful.

Now let’s try with a white background:

  • Edit → Background Colour → White
  • {this is good for making figures, but you will need a very pastel map colour (not so dark)}
  • To “highlight” a residue:
  • Draw → Additional Representation…
  • Ball & Stick → Add Representation
  • Use the Display Manager’s check buttons to undisplay the ligand when done.

2: Navigation

{Note: When I say “G” I mean just “G” not “Shift G” - I refer to the symbol on the keycap, not the letter that appears if one was typing text (and this applies to any letter, not just “G”)}

Moving around the molecule:

  • Middle-mouse click over and atom to centre the view on that atom
  • Left-mouse Double-click to toggle the atom label
  • “Space” is forwards along the chain
  • “Shift Space” is backwards backwards along the chain

To make Coot understand that it is from this residue from which we wish to move forwards (or backwards):

  • press the “P” key)

To drag-pan the view:

  • Middle-mouse click and drag {just like PyMOL}

To change the “residue to residue” speed:

  • Edit → Preferences…
  • Smooth Recentering → 10 steps {smaller numbers are quicker}

If you don’t have a fast computer this can help:

  • in the left-hand vertical bar, press the “Maps” button
  • Select the “Dragged Map” tab
  • “Active Map on Dragging?” → “No”
  • OK

You might like the residue reorienting mode:

  • Calculate → Scripting → Python
  • set_reorienting_next_residue_mode(1)
  • Now the main-chain orientation of the next residue will rotate to match the current one - that might take a bit of getting used to, so you can turn it off:
  • set_reorienting_next_residue_mode(0)

3: Short-cuts and Interface

To go to a residue somewhere in your structure:

  • “Ctrl G” {see the little dialog?}
  • Type the residue number (and the chain-ID, if needed)
  • Press Enter {Coot recentres on the specified residue}
  • The recentering also works with 3 single-letter-code letters, e.g.HEY (it only works for the first one)

You can toggle spin and rock the view with: - “I” (as in “indigo”), “Ctrl R” {note that “I” is distinct from “Shift I” - that’s something else}

If coot goes unresponsive, doesn’t seem to respond to clicks and mouse movement, then there’s a good chance that you have started a “Ctrl-G” dialog, but not typed “Return”. If that’s the case simply press “Esc” (escape).

3.1 Install a Few Buttons

  • Right-mouse click on the right-hand side of the horizontal tool-button bar
  • Select “Manage Buttons (add, delete Buttons)”
  • Add “Sphere Refine +”, “Tandem Refine” and “Backrub Rotamers”
  • Toggle on the “Backrub Rotamers” button {the button will turn pink}

3.2 Install Key-bindings

  • Edit → Settings → Install Template Key-bindings

To see those key-bindings at a later date:

  • Edit → Settings → Key-Bindings {see what the keybindings are}

3.3 Install a Few Extensions

  • File → Curlew
  • From there, select and install “Black Box Morph and Fit”, “Chain Refine”, “Morph” “Refinement Tools”, “Dynamic Validation” and “Expand Map Radius”

3.4 Try it out

Use the cursor to navigate using the map

  • “G” {brings the blob under the cursor to the centre of the screen}

To undo last navigation/screen recentre:

  • “U”

Also:

  • ”+” and “-“ Change the contour level
  • “M” and “N” for zoom in and out
  • “D” and “F” for depth of field/clipping planes, increase and decrease

3.5 Quick Save-As

The new version of coot is a lot more advanced and a bit more crashy than the standard version. It’s a good idea to save every now and then

  • making it easier to recover.

    • “Ctrl S” {for quick-save-as}

    Quick Save as will save all models that have not been saved and will save the session too.

    (This is for your notes, don’t act on this: if you wish to run/execute/evaluate your saved session file then you can do that using Calculate → Run Script…)

4: Model-Building

4.1 Find and Fix a Bad Rotamer

  • Validate → Density Fit Analysis…
  • Click on the bar for residue A89 You might need to reset the weight: 2.0 seems like a good number

Fix the Rotamer, First the Traditional usage

  • the vertical toolbar can display labels also (if you like):
  • Click on the green triangle arrow at the bottom of the vertical toolbar:
  • Click Icons and Text
  • click on Auto-Fit Rotamer in the vertical tool-bar

But let’s use the Key-bindings to speeds up, so undo the previous fit:

  • “Ctrl Z”
  • “J” {auto-fit rotamer}

And to tidy up after that:

  • “X” {does real-space refinement}

4.2 Let’s Find Something Else to Fix

Validate → Ramachandran Plot

  • click on “Outliers Only” - click on Residue A41

Note: Peptide Flags: - Green: cis-peptide in front of a PRO - Yellow: twisted trans-peptide - Red: cis-peptide in front of a non-PRO

When it comes to fixing up model-building/validation problems, often times “Neighbour Refine” is the first tool to try. If we try that, we see that it doesn’t work in this case. We need to look and think. We see that the one of the peptides is facing the wrong way and we need to flip it:

So, let’s fix this with Real Space Refine and flipping:

  • “H” {does a neighbour Refine}
  • “Q” {does a pep-flip}
  • “H” {tidy up with a RSR}

5: Now To the Demo Box!

The point of this exercise is for you to see/learn how Coot behaves with lower resolution data.

Use the Display Manager to delete all the Maps and Models.

For these refinements, it might be useful to use the Coot atom interaction dots:

Refine → Contact Dots On

OK, let’s find the script and run it:

  • Calculate → Run Script → demo-box-of-buttons-madrid.scm
  • Crankshaft Peptide {brings the problem to the centre of the screen}
  • Take a look at the problem…
    • {Yikes!}
  • Morph → Crankshaft Peptide Optimizer
  • Does it fit better now?
  • Sphere Refine
    • {things improve, balls are green}
    • OK

Now let’s try something more tricky:

Use the Display Manager (again) to delete all the Maps and Models.

In the Demo Box dialog:

  • Wonky N-terminus
  • Rotamer Markup On
  • Centre on Residue A4
  • Tandem Refine
  • {Play with it} - make the balls go green and remove the clashes

Note: the colour of the balls and the dodecahedra represent the probability of the Ramachandran Plot and the Rotamer for each residues respectively. Rich green is high probability and rich red is very low. Generally speaking you should be aiming for various shades of green but have the expectation that you won’t always get there (especially for rotamers).

Now try that with a blurred map:

  • Undo
  • {the first “Undo” gives us a dialog to select the molecule, we need to “Undo” again to actually activate the Undo (this is a bad interface, sorry)}
  • Calculate → Map Sharpen and Blur…
  • Blur to about 60 or 70Ų {to resemble cryo-EM map}
  • Tandem Refine again