e2boxer.py manual picking
Home > Picking particles > Manual picking using e2boxer.py
###Manual particle picking with EMAN2: e2boxer.py
###Launching e2boxer.py
Manual picking with e2boxer.py is straightforward & intuitive due to its graphical user interface (GUI).
To open a micrograph, simply provide the micrograph name alongside the e2boxer.py command:
$ e2boxer.py Frame23.mrc
This will open three windows:
- A window containing your micrograph (left window below)
- A blank window that will show picked particles (middle window below)
- Main menu window (right window below)
First, you must select 'manual' from the Main menu window along with a diameter (in pixels) for your sample. To learn more about how to measure the diameter of your sample & picking a box size, read more here:
###Picking particles
Within the micrograph, start clicking each particle that you would like to extract. White boxes will appear around them:
Continue picking particles until you have picked everything that you want.
Tips for using EMAN2:
- To change the zoom on the micrograph, simply scroll your mouse wheel OR right click image and adjust 'magnification.'
- To remove particles that you don't want, you can press the 'Clear' button in the menu to remove all particle picks.
###Saving particles
When you are ready to save your particles, you have two choices for the output format from EMAN2:
- Extracting particle stacks
- Coordinate files (e.g. .box) containing the X & Y coordinates for each particle within the micrograph
To save your particles, click the 'Write Output' button within the Main menu window:
This will open a new window. You must perform the following actions:
- Select the micrograph from which you will extract/save particles (micrograph(s) that are highlighted are 'selected')
- Specify if the output is a particle stack or particle X & Y coordinates
- Specify the output box size
- Specify the output particle stack format (if applicable)
- Specify the type of image normalization (read about normalization here)
- Specify output suffix
####Saving output as particle stack
You can save your particles directly into a particle stack that is .img/hed, .spi, or other formats (read more about file formats here).
#####Output location This particle stack will be within a directory below where your micrograph is located. If you micrograph is located in /home/micros, then your stack will be in directory /home/micros/particles named with the same base name as the micrograph.
Pros:
- This method allows you to extract directly your particles that you just picked.
Cons:
- Manual picking is cumbersome, and you'll have to redo these steps for each micrograph in your dataset. Semi-automated and automated particle picking is usually preferred.
####Saving output as particle coordinate file (.box)
Instead of saving the particle stack, you can save the X & Y coordinates of the particles you just picked in a text file with the .box extension. By saving .box files for all of your micrographs, you can extract your particles into a single stack using a single command.
#####Output location
This coordinate file will be within the same directory as your micrographs. If your micrographs are located in /home/micros, then your coordinate file will be in directory /home/micros named with the same base name as the micrograph and the extension of .box.
Pros:
- This method allows you to using the makeStack.py command to extract all of your particles from multiple micrographs simultaneously, while also giving the option to perform CTF correction of the extracted particles.
Cons:
- You will only get coordinate files as outputs, so you can't see your particle stack until later when you extract all of your data.