Tutorial

Overview

HELIS integrates several tools to assist in analyzing helical structures in Cryo-ET data. With this software, you can pick filaments from a given tomogram, obtain their cylindrical projections, determine the corresponding helical parameters, and perform single filament sub-tomogram averaging (SF-STA).

Load Data

Step 0: Get the tutorial dataset

In this tutorial, we will use the tomogram provided in EMD-15627. Please download and unzip the dataset before proceeding.

Step 1: Load Tomogram data

Click the Tomo button at the top-left of the GUI. In the pop-up dialog, select the downloaded tomogram file. After loading, the GUI will look like this:

Pick Filament

Step 1: Scan the tomogram

Use the up and down arrows of the Layer idx control or edit its value directly to navigate through the tomogram layers. Move to the 41st layer for this tutorial.

Step 2: Picking the filament manually

In the 41st layer, we can find a tube with a sharp turn. To avoid issues at the turning point, use only the upper half of the tube.

1. Click the Add Filament button to enter filament-picking mode.
2. Click three points along the target tube (as shown below).
3. Click the Accept button to save the selected points as a filament.

Step 3: Check the filament

After successfully adding a new filament, an item named Filament 1 will appear in the Filaments list box. Click this item to display a blue line representing the filament in the tomogram view.

Tune Filament

Step 1: Entering Filament Tuning Mode

With Filament 1 selected, the Tune Filament button will become active. Click it to begin tuning the filament.

Step 2: Switch to Filament View

Switch to the Filament tab. You will see three views: the cylindrical projection, filament sections, and the spectrum.

Step 3: Determine Filament Tracing Threshold

1. In the Filament tab, set the inner and outer radius values to 8 and 14, respectively. This adjustment matches the tube shape and improves tracing accuracy.
2. Switch back to the Tomogram tab for coarse selection.
3. Click the Estimate Thres button. After a few seconds, the thres field will be filled with an auto-estimated value (e.g., 0.0357 in this case).

Step 4: Tracing Full Filament

1. Set the thres value to 0.05 (slightly higher than the estimated value for a more conservative tracing).
2. Click the Trace button to automatically extend the filament in both directions.

Scan through the tomogram to verify that the tracing aligns with the real tube. If some points are misaligned:

1. Use the Next Pt or Prev Pt buttons to select the problematic points.
2. Click the Rmv Pt button to remove them.
3. Click the Update button to save the tracing results.

Step 5: Auto recenter

1. Click the Auto Cen button to automatically recenter the filament.
2. Click the Update button to save the recentering results.

These steps can be repeated as needed.

Step 6: Final Adjustment

For fine-tuning:

1. Switch to the Filament tab.
2. Use the Next Pt and Prev Pt buttons to navigate sample points. The filament section figure will help verify if the sample points are centered.
3. If a point is off-center, click the Mov Pt button and select the correct position in the filament section view.

Once all points are centralized, click the Update button to re-unroll the filament for further analysis. The final GUI should resemble this:

In this view, the cylindrical projection is uniform along the z-axis, and the tubes in the filament section view are centered.

Step 7: Save Filament Information

1. Click the Finish Tuning button to exit tuning mode.
2. Click the Save button to save the filament information file for future use.

Determine Helical Parameters

Step 1: Spectrum Modification

If you exited tuning mode earlier, click the Tune Filament button to re-enter.

1. Set the Gamma value to 12 to suppress noise and highlight diffraction points in the spectrum.
2. Click the Rmv Bg button to remove the spectrum background.

The modified spectrum will appear as follows:

Step 2: Manually Set Reciprocal Primitive Vectors

1. Click Set Va and then select a diffraction point in the spectrum.
2. Click Set Vb and select another diffraction point.

Ensure that the grid formed by these vectors covers all diffraction points.

Step 3: Automatically Optimization

Click the Opt Vs button to optimize the manually selected vectors. The reciprocal vector coordinates and corresponding helical parameters will be displayed in the bottom-right section of the GUI.

Step 4: Symmetrical Averaging

1. Click the Avg Surf button to average the cylindrical projection based on the helical symmetry.
2. Click the SF-STA button to segment and align the helical structure using the helical parameters.