Difference between revisions of "Pipelines for Workshop Heidelberg 2018"
Line 39: | Line 39: | ||
The initial structure of the folder contains two items | The initial structure of the folder contains two items | ||
− | <pre> ls work/1 | + | <pre>ls work/1 |
rawStack.mrc | rawStack.mrc | ||
nominalTilts.tlt | nominalTilts.tlt | ||
Line 91: | Line 91: | ||
== Management of tomogram data == | == Management of tomogram data == | ||
− | == Subtomogram averaging | + | |
+ | == Subtomogram averaging == |
Revision as of 00:06, 22 August 2018
These materials describe the procedural steps required for high resolution tomography.
Contents
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.
Material organization
The data corresponding to each tilt series is under the folders work/1 and work/2. Intermediate results corresponding to each tilt series will be stored in the corresponding folders.
The initial structure of the folder contains two items
ls work/1 rawStack.mrc nominalTilts.tlt
The additional processing steps will gradually generate more elements pert tilt.
Copying data to your account
You will need to copy the provided data into you account:
cp -r /g/embl/workshop/teach11/data/work .
Matlab environment
Matlab provides a convenient syntax for manipulation of sets of paths.
Preprocessing
Each of the preprocessing steps can be computed separately for each tilt series. We have thus provided a set of functions, each one a wrapper that accepts as input one index that identifies a tilt series and creates an output with the right naming convention. They are intended to be callable on a loop like:
for i=1:2 emboFunction('work',i); end
If new tilt series are added to increase resolution, you just need to apply the function on additional indices.
defocus estimation
The function emboDefocusEstimation will run ctffind4 on the input data, creating the following items
- Input:
- Output:
tilt series alignment
- Input:
- Output:
dose filtering
- Input:
- Output:
CTF correction of tilt series
- Input:
- Output:
Reconstruction of tomogram
- Input:
- Output: