Pipelines for Workshop Heidelberg 2018

These materials describe the procedural steps required for high resolution tomography.

Pipeline

Input data

We will start with two tilt series. A drift correction has already been applied on each tilt using motioncorr2, so that each micrograph in a tilt series is an averaged movie. The pixel size is 2.7A (the tilt series have been binned by fourier cropping to ease the computation ), and the structures of interest form spherical lattices.

Procedural steps

Preprocessing steps

As we will initially work with already drift-corrected tilt series,

• Defocus estimation with ctffind4
• Alignment of tilt series with Dynamo
• Exposure filter on the aligned tilt series.
• Stripe-based CTF correction (with ctfphaseflip from Imod)
• Creation of tomograms with Dynamo

These preprocessing operations that go from the tilt series to the tomograms can be used for any sample.

Tomogram management

This part is specific to the particular geometry of the proteins of interest.

• Cataloguing the tomograms (archiving)
• Annotation of vesicle positions.
• Extraction of putative particles based on the vesicle positions.
• Ellimination of repeated particles.

Subtomogram averaging

• Extraction of true particles.
• Splitting in odd and even data sets.
• Independent alignment and resolution determination.