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• How do I use e2boxer to automatically box many images (potentially hundreds) from the command line?

• What are Dynapix and Anchor for?

• Is there an iterative centration option like in boxer?

• The boxes around the particles can be quite colorful white, green, yellow, black; can you give me a translation to this color coding?

• Why is it creating all those *.box.bak files ? Are they important?

• What is the plot in the advanced tab?

• What is a peak profile?

• How do I deal with bad particles and ice contamination?

• How do I export particles files that have been saved in database format?

Q: How do I use e2boxer to automatically box many images (potentially hundreds) from the command line?

A: As of July 22nd 2008 the answer is

First load up 3-10 images in the interface and use the tools to do autoboxing. Once everything looks fine and the automated boxing is behaving nicely in the interface click 'done' - this will save the autoboxing parameters to a local database.

Then quit to the command line and run something like this (make sure you specify the output you want)

e2boxer.py *.mrc --auto=db --write_coord_files --write_box_images

What's really happening is the contents of the database are being examined to autobox all of the images. When you do use the --auto=db option from the command line it uses the most recently modified/created/used autoboxer in the interface.

Some people may have both near-to and far-from focus images and may be concerned about the results of autoboxing using a single set of autoboxing parameters. At the moment there is no quick work around for this situation because you can only use one autoboxer from the database to box many images automatically from the command line - you have no choice over which 'autoboxer instance' you're using either, it is simply the most recently created/modified/used autoboxer. If you want to break your images up in close to focus and far from focus groups you'd have to put them in separate directories. But it wouldn't be surprising if you could make a single autoboxer that worked well on both close to and far from focus images- i.e. it may not be necessary to break them up at all.

Q: What are Dynapix and Anchor for ?

A: As of June 20th 2008 the answer is

• Dynapix is a mode of operation where e2boxer does automatic picking in real time based on what 'references' the user adds (or deletes, or moves).
• The presence of the anchoring options is debated - it means that a reference added is not used to refresh the correlation image - instead it is only used to refresh the automatic picking parameters. That may sound confusing - for the time being you could probably ignore it, or you could try and see what happens.

Q: Is there an iterative centration option like in boxer ?

A: As of June 20th 2008 the answer is

e2boxer does its own centering by default. Currently the user has no control over it. This could change if people demand it. So far most people are happy with the centering... The details are e2boxer does autoboxing in shrunken images but then automatically recenters the boxed particle in the big (original) image using translational (integer only) alignment.

Q: The boxes around the particles can be quite colorful white, green, yellow, black; can you give me a translation to this color coding ?

A: As of June 20th 2008 the answer is

• green - an automatically selected box
• white - a manually specified box
• black - a manually specified box that is being used as a reference for autoboxing
• yellow - the currently selected box, for visual reference only, there are some bugs associated with this color which need fixing

Q: Why is it creating all those *.box.bak files ? Are they important?

A: The '.bak' stands for backup - when you tell e2boxer to write a .box coordinate file it will automatically create the name of the output file based on the image name, and sometimes this file already exists. If it does exist it's copied and written as the '.box.bak' file, also a time stamp is included in the new file name. You can get around this by ticking the 'force' tick box in the 'Output' section in the main controller.

Q: What is the plot in the advanced tab?

A: Very broadly speaking, the plot displays an optimum correlation 'peak profile' (that is upside down). This peak profile is generated automatically using the references you have specified in the interface - specifically the local correlation peaks nearest to the references are located and the information in the local neighborhoods is condensed into single 'correlation peak profiles'. In the plot the horizontal axis represents radius, and the vertical axis is representative of pixel value ratio (relative to the local correlation maximum score). All of the peak profiles of each of the individual references are carefully condensed into an optimum profile, and this is what you see in the advanced tab. When you have 'selective' chosen the autoboxing algorithm uses two pieces of information to autobox - a correlation threshold, and also the part of the optimum profile that corresponds to the largest drop (greatest in the vertical direction). This latter parameter is shown by a vertical green bar in the plot in the advanced tab. If you choose 'more selective', then the autoboxer uses all of the optimum profile up to and including the radius that corresponds to the largest drop (in addition to the correlation threshold).

Q What is a peak profile?

A The pixels around the correlation peak are broken up into radial segments based on distance from the peak. The distance is rounded and then defines which radial segment a certain pixel belongs to. That's the first part. The second part is finding the largest pixel in each of the radial segments. So then we have one pixel for every radial segment. And this defines a kind of radial profile of the peak. To make things relative the profile is normalized to the maxima itself (the peak correlation pixel), either by dividing or by taking the difference. We prefer dividing at the moment, but users can change this in the interface in the advanced tab.

You can also see the peak profile used to autobox in the advanced tab in e2boxer. It's just a 1D plot. We automatically select other maxima depending on whether their profiles are under or above this plot (we use above). By default we just compare using a single part (one radial value) of the peak profile, that radius corresponding to the largest difference, and this is the selective mode. In the "more selective" mode the peak profile up to and including the "largest difference radius" is used to auto select other maxima.

Q How do I deal with bad particles and ice contamination?

A Hold shift and left click on any box to delete it. You can also hold shift, hold left click down, and drag your cursor around to delete multiple boxes. To deal with large areas of ice contamination use the erase option. Click on erase and then left click and drag to erase large areas. Hold down shift and use the mouse wheel to change the size of the erased area, or set the size of the erased area in the main inspector widget.

Q How do I export particles files that have been saved in database format?

A There are currently two ways to convert particles from bdb format to img (or any other). The easiest way is to use e2display.py - Just run this script with no arguments - you should see the EM browser pop up. Then browse to your image files and right click on them. If they were saved by e2boxer they are in the particles directory. You should see a menu pop up that has a Save As option. You can select more than one file to "Save As" and the program will run a loop asking you to name each file as it saves them.

They way to export them with e2proc2d.py us using an argument like this:

e2proc2d.py bdb:particles#image1 image1.img

You see there is no need for the .bdb, and also that accessing images in bdb format requires a special syntax.