Introduction

In this tutorial, we will reprocess a five tilt-series subset of EMPIAR-10164, a cryo-ET dataset of HIV-1 virus like particles (VLPs) from the Briggs group.

Data will be processed ab initio, highlighting many important concepts and exposing you to powerful tools which will help with your own data processing needs.

Note

This subset was used to benchmark both NovaCTF and Warp.

This processing will be carried out combining the Warp-RELION-M pipeline with tools from Dynamo. Combination of these tools yields a powerful, flexible framework for ab initio and geometrical approaches to subtomogram averaging, benefitting from multi-particle refinement in M.

From these five tilt-series, this procedure can produce a 3.4 Å reconstruction of the HIV-1 CA-SP1 hexamer.

A note about scripting

As the software ecosystem for cryo-ET matures, some scripting is often required to achieve your goals. This tutorial is provided with all necessary scripts.

For illustrative purposes, the contents of these scripts will appear as code blocks in the tutorial when used.

% this is a comment
disp('Hello World!')

Reading these blocks is intended to serve as a gentle introduction to MATLAB scripting for working with the powerful tools Dynamo has to offer.

For general purpose scripting, we personally prefer Python for its wealth of open source data science infrastructure and educational resources.

Requirements and Setup

  • A reasonably modern CPU.

  • At least 32 GB RAM.

  • At least 1 NVIDIA GPU, with at least 8 GB of dedicated memory.

  • At least 10 GB of free disk space.

More RAM will be required for particle extraction at the smallest pixel sizes; we used a machine with 128GB. To keep read/write times low, we recommend a Solid State Drive (SSD).

  • Access to both Windows and Linux operating systems

  • MATLAB r2019a or later with the following toolboxes installed

    • Image Processing Toolbox

    • Parallel Computing Toolbox

    • Computer Vision Toolbox

    • Curve Fitting Toolbox

  • IMOD

  • Warp v1.0.9 or later

  • Relion 3.0.8 or later

  • Dynamo 1.1.478 or later

  • dynamo2m

  • autoalign_dynamo

  • scripts provided with this guide

If you need help with setting up any of these tools, follow the link to the respective documentation, or check out the software packages and computing sections.