Chimerax filaments

Tracing filaments in ChimeraX

Filaments such as microtubules or actin are annotated by their centreline: an ordered curve through the middle of the filament, one per filament, each with its own ID. ChimeraX-copick traces them directly on the tomogram: you click control points along a filament and copick draws a smooth curve through them. The same tools cut, join, reverse and delete filaments, which makes them just as useful for curating automatic traces, for example those of copick convert seg2fil.

This tutorial uses the microtubule project from Tracing microtubules with easymode and copick-utils, but any copick project with a tomogram works. It assumes you know how to open a project and a tomogram in ChimeraX-copick; if not, start with the ChimeraX-copick tutorial.

Step 0: Prerequisites

Filament tracing needs ChimeraX-copick 1.15 or later, which brings copick ≥ 1.28. Install or update it from the ChimeraX command line:

toolshed install copick

Step 1: Declare a filament object

Only objects declared as filaments can be traced. A filament object is a particle object with a filament specification: its radius is the tube radius, and its polarity says whether the filament has a direction. If your project does not have one yet, click ✏️ Edit Object Types in the copick panel, select the object (or Add New), tick Is Particle and Is Filament, and set the radius and polarity under Filament Properties:

From the command line, the same object is created with:

copick add object -c config.json \
  --name microtubule --object-type filament --radius 120 --polar

Step 2: Open a tomogram and the Copick Edit toolbar

Start copick with copick start config.json and open a tomogram, for example run 35962. All filament tools live on the Copick Edit tab of the toolbar:

Step 3: Start a new filament set

Click New Filaments (or 📄 in the Filaments tab of the copick panel). The dialog lists only filament objects; choose a user and a session for the new set:

Start tracing creates the set, switches the right mouse button to trace mode and opens the Copick Annotate window next to the ArtiaX options. The label at the top of the view always shows what the right mouse button does.

Step 4: Trace

Find a filament on the tomogram plane and right-click along it to place control points; the centreline is a smooth (Catmull-Rom) curve through them. Move through the tomogram with Shift + mouse wheel to follow a filament that leaves the plane: new points are placed on whatever plane is shown, including orthoplanes and tilted slabs.

• Move a point: right-click a control point and drag it. The whole drag is one step to undo.
• Remove a point: Shift + right-click it.
• Where new points go: the New points menu in the Copick Annotate window adds them at the end (default), at the start, or between nearest, which inserts a point into the closest segment, for example to refine a curve.
• Start the next filament: New in the toolbar, ＋ in the Annotate window, or N+F.

While you trace, the control points of the active filament are white, and those of the other filaments take their filament's colour. Arrowheads on each tube show the filament's direction, from its first to its last point. For a polar filament such as a microtubule, use Reverse (R+V) if it points the wrong way.

Step 5: Join, cut and reverse

Join connects filaments end to end, for example two pieces of one microtubule that you traced separately or that a segmentation broke apart. Select the filaments, either as rows in the Annotate window (Ctrl / Shift + click) or by Ctrl + clicking their tubes in the view (Shift + Ctrl + click adds to the selection); the two stay in sync. Then click ⧉ in the Annotate window, Join in the toolbar, or press J+F. Copick connects the nearest ends, reversing a piece if needed, and the joined filament keeps the ID and direction of the current one.

Cut does the opposite. Switch to cut mode with Cut (toolbar or Annotate window) or C+F, and right-click a filament where it should be split. The first piece keeps the filament's ID, the second gets the next free ID, and both keep its direction. Switch back to trace mode with Trace or T+F.

Step 6: Browse the filaments

The Filaments page of the Copick Annotate window lists the filaments of the active set with their length and score. Click the list header to sort by ID, length or score; the filter box takes IDs (1-5, 9) or text. ◀ ▶ step through the filaments, 🎯 (or a double-click) centres the view on one, and the checkboxes show or hide them. ⇄ reverses the current filament and 🗑 deletes the selected ones.

Step 7: Save

Click 💾 in the Annotate window (or Save in the Filaments group of the toolbar) to save the active set. Optionally, copick also writes picks sampled along the filaments at a fixed spacing, oriented along each filament and carrying its ID, for example every 82 Å (one tubulin dimer) for subtomogram averaging:

Step 8: Curate automatic traces

Filament sets from copick convert seg2fil open like any other: double-click microtubule:seg2fil/1 in the Filaments tab and click it to make it active. The traces are Catmull-Rom curves, so their control points can be added, moved and removed like those of a filament traced by hand, and cut and join work as above. Save the result under a new session so the automatic traces stay as they are.

Filaments stored as B-splines (seg2fil --curve bspline, or traces from copick-utils 1.9) can be moved and cut but not edited point by point: convert one with → Catmull-Rom in the Annotate window first.

Quick reference