Doppio STA Tutorial

Introduction

Welcome to the Doppio Tomo Tutorial!

This tutorial will guide you through the basics of using Doppio Tomo for your projects.

Contents:

What is Doppio Tomo?

Doppio Tomo is a web-based tool designed to simplify RELION processes, offering:

Ease of use: Intuitive design for beginners.
Flexibility: Adaptable to different workflows.
Performance: Optimized metadata handling for reliability.

Doppio has pipeliner plugins for each step in RELION tomography. The following table shows those tomography specific plugins and their equivalent RELION job tools.

Pipeliner job	RELION job
relion.importtomo	Import
relion.importtomo.coordinates	Import coordinates
relion.motioncorr.tomo.own	Motion correction (own)
relion.ctffind.tomo.ctffind4	CTF estimation
relion.excludetilts	Exclude tilt-images
relion.aligntiltseries.imod_fiducial	Align tilt-series (IMOD fiducial)
relion.aligntiltseries.imod_patchtrack	Align tilt-series (IMOD patchtrack)
relion.aligntiltseries.aretomo	Align tilt-series (AreTomo2)
relion.reconstructtomograms	Reconstruct tomograms
relion.reconstructparticletomo	Reconstruct particle
relion.denoisetomo	Denoise tomograms (CryoCARE: Predict)
relion.denoisetomo.train	Denoise tomograms (CryoCARE: Train)
relion.picktomo	Pick tomograms
relion.pseudosubtomo	Extract subtomos
relion.initialmodel.tomo	3D initial reference
relion.class3d.tomo	3D classification
relion.refine3d.tomo	3D auto-refine
relion.refine3d.tomo.helical	3D auto-refine (helical)
relion.ctfrefinetomo	CTF refinement
relion.framealigntomo	Bayesian polishing

There are a number of common jobs between tomography and single-particle. Those are not listed in the above table.

Example image — Example tomographic data processing workflow

Scope of the tutorial

In this tutorial, you will learn how to:

Set up and manage projects within Doppio.
Use Doppio tomography tools for processing cryo-electron tomographic data.

The steps in this tutorial are similar to RELION Subtomogram Average tutorial. Please refer to RELION STA tutorial for more information.

Prerequisites

To get the most of the pipeline you need following software packages installed on your PATH

IMOD
AreTomo
MotionCor2
CTFFind4
CryoCARE

For installation instructions, see the installation section.

Let’s get started!

Doppio user guide https://www.ccpem.ac.uk/docs/doppio/user_guide.html covers many useful aspects of using Doppio.

Note

In this tutorial, we will cover Doppio for tomographic data processing only.

This tutorial will walk you through the tomography pipeline of relion-5.0 using Doppio tools. We will use immature HIV-1 dMACANC VLPs (EMPIAR-10164) dataset and use only 2 tilt series TS_01 and TS_03.

To start processing we need to create a project folder and let’s name it STA_tutorial. We’ll do everything related to this project in this folder. Indside the project folder, let’s create another two subfolders frames and mdoc.

mkdir STA_tutorial               # This is the project folder
cd STA_tutorial
mkdir frames mdoc                # Sub-folders for frames and mdoc files

The frames folder will contain the raw tilt series data (movies, or individual motion-corrected images) and the mdoc folder will contain the mdoc files containing metadata for each tilt series. The data set is available at EMPIAR-10164 https://www.ebi.ac.uk/empiar/EMPIAR-10164/. The data set contains several tilt series of immature HIV-1 dMACANC VLPs, which were collected on a Titan Krios microscope at 300 kV with a Gatan K3 camera. Download TS_01 and TS_03 tilt series, and the corresponding mdoc files. Make sure all the raw movies/images go into frames folder and mdoc files go into mdoc folder.

Starting Doppio and changing to tomography theme

Open the terminal and type doppio_web to start the Doppio GUI. This will open a new tab in your web browser with the Doppio GUI. The Doppio default theme is green. It includes all the tools. Doppio tomography theme is purple and it includes only the tools needed for tomography processing. To change the theme to tomography, first click on the small cogwheel icon on the top left corner of the GUI to access the Doppio settings and then click on the EDIT button and choose the tomography.json file from the themes and click SAVE.

Setting the project directory path in Doppio

To set the project path in Doppio, click on the ‘pen’ icon on the top left corner of the GUI and click on it to edit the project path. Please type the path to the project folder you created above and then click on the Tick icon to register the path.

Next, we will import data into the pipeline.