Warping
Warping
Ensure that all of your micrographs are in the same directory.
If you want to use a default picking algorithm:
Simply select your micrograph directory in the Warp interface, and modify the microscope voltage, dose etc. accordingly and hit ‘Start Processing’. Please consult the Warp documentation for detailed information.
Training
- Create a temporary subfolder with ~20 micrographs, and run warp on this subset. A good starting point for small particles is the vertex picking routine; try one of the bev sets for virus samples on lacey grids; rota for large virus samples.
- Once this has run, selecting/deselecting good/bad particles accordingly using the ctrl key.
- For lacey grids, it is often helpful to use the highlight tool to demarcate carbon areas so that warp avoids picking particles in these regions.
- When you are happy, stop the run. You are now ready to re-train your picking algorithm. To do this, select the particle picking procedure on the left-hand pane, and select ‘train’. This will then prompt you to give a wild card of the good particles (should be in your matching folder). Select this, before selecting train-note that warp will just crash if you do not set the good examples.
Output
Warp will write out processed data to the following folders:
Average Motion-corrected micrographs.
Matching Coordinate star files.
Extract Extracted particles.
RUNNING RELION WITH WARP COORDINATES
- Copy warp coodinates over to your relion working directory. Coordinates should be located in <pathtowarpprocessing>/matching/*.star . To avoid copying over the 'false positives', ensure that the wild card for the star file includes *<pickingname>.star
cd relion-wd
mkdir micrographs
cp /data/bowmore/krios/bowden/vicky/awesome-data/matching/*propicked.star micrographs/.
It is actually cleaner to copy your coordinates over to whichever directory you have sym-linked your micrographs to.
- Change the coordinate star file names to adhere to relion's expectations.
cd coordinates
rename propicked _pick *.star
- Remove spurious headers from warp using custom scripts. Moan to HelenD if this fails.
sh /well/strubi/hmed/apps/microscopy/convertwarpcoords/runremove.sh
- This should remove the spurious headers from offending files. To avoid screwing up, some 'old' star files. Move these out of the way into a temporary directory for the time being.
mkdir old
mv old*star old/.
- Now you should be ready to import with relion. Open the relion gui, navigate to the import tab, and enter the file path to your coordinates into the appropriate window. In the dropdown menu, select 'Import 2D/3D coordinates'.
coordinates/*_pick.star
- Check that the data has been successfully imported by going into the Import directory and corresponding job number. There should be a star file named according to the provided suffix, and a fold with your coordinate directory (in this case 'coordinates') that contains all of your coordinate star files (yes, another copy of them->#relion_efficiency).
- At this stage, it is likely that your coordinate path does not correspond to the micrograph path. There are two ways to avoid this. (a) Post-hackfix-emulate the micrograph file directory in your Import/job00x/coordinates i.e. Import/job00x/<path-to-micrographs> . (b) Start from number 1, and copy coordinates over to your micrographs directory. We did (a) first time round.
- Run extraction! Open the relion gui, and run extract with your Import/job00x/coordinates_pick.star files and micrographs_ctf.star micrographs. This should should use the dose-weighted micrographs that are listed in the micrographs star file.