frealix usage

Hi Liz,

Thanks for your interest in Frealix. Indeed in at least some cases, Frealix can refine Frealign alignment parameters for filaments and improve the final resolution, so definitely worth a try.

Your goal initially will be to create a first waypoints.ali file, which is where Frealix stores alignment parameters for each waypoint of each filament. As things stand, the conversion from Frealign to Frealix is not handled by flx_wrap.rb, unfortunately (it'll only do startup from eman boxer or IMOD coordinates for now).

Instead, I'd recommend either you write an awk-based script to do things manually, or you use frealix mode frealign_stack_to_frealix_ali. In Frealix 1.1, this should handle conversion from frealign v8 correctly, though it will take a long time to run. If you used Frealign v9, I will need to send you my development version of Frealix 1.2. Let me know.

To use this mode, first create a text file (conversion.txt, say) with the following content edited appropriately:

frealix_mode 			frealign_stack_to_frealix_ali
frealign_par_filename		/path/to/frealign.par			# Frealign parameter file.
stack_filename			/path/to/stack/of/binned/particles.mrc	# Stack of particles that were processed by frealign
film_filenames			/path/to/your/micrographs/mic_???.mrc	# The question marks represent [0-9] digits and indicate the micrograph number.
film_selection			1-100,103-120,124,126			# Which film numbers to look for. Comma-separated list, where - indicates contiguous range.
list_of_film_numbers		film_number_for_each_particle.txt	# A text file with number of lines = number of particles in input stack. On each line, give the number of the micrograph from which the particle was extracted.
ali_filename			waypoints_from_frealign.ali		# Filename for output waypoints file
pixel_size			5.43					# In Angstroms, the pixel size of the stack of particle images and of the micrographs
check_180_rotation		f					# Set this to t if some of your particle images were rotated by 180 degrees

You will then run the following command to run the conversion:

/path/to/frealix conversion.txt

The issue when converting from frealign to frealix is that the frealign parameter file does not keep track of which micrograph each particle was extracted from nor what the particle coordinates are within that micrograph. To recover this information, mode frealign_stack_to_frealix_ali reads in each particle image at a time and literally looks for it in the micrographs (doing something like automatic particle picking). If you give it the "list_of_film_numbers" parameter described above, you save it a lot of time, because you're telling it in which micrograph it should expect to find each particle. Unfortunately, this feature is not available in Frealix 1.1. If this information is not available (or if you're running Frealix 1.1), it will look for each particle in each micrograph which can take a long time if you're dealing with a large dataset.

Regardless of which version of Frealix you use, you should run this with a binned / resampled stack of particles and micrographs, with a pixel size of, say, 5 to 10 Angstroms. (Slight errors in X,Y do not matter much anyway, and can usually be recovered in the first round of refinement).

Once it has found each particle and it now knows micrograph it belongs to and its coordinates, it writes out a waypoints.ali file which you can use as a starting point for frealix refinement.

You would then use flx_wrap.rb to start a Frealix refinement, by pointing it to this waypoints.ali file and to your existing reconstruction (or a low-pass version thereof).

Regarding naming conventions: the waypoints.ali file specifies on which micrograph each filament is located. When Frealix needs to access the image of that filament, it will look for the micrograph image by taking the filename pattern you supply, and substituting the micrograph id number into the part of the pattern that contains question marks. If you supplied the filename pattern given in the above example, and it's looking for a filament on micrograph 12, it would expect to find a file named /path/to/your/micrographs/mic_012.mrc. Hope this makes sense.

In the example you supplied (my_micrographs/14oct28a_0????sq_v01_0????hl_0????en-a.mrc), Frealix substituted the micrograph number into the first stretch of qmarks it found and left the other qmarks alone. So the solution in your case, it to simplify the naming scheme of your micrographs. Perhaps you could create a new directory with symbolic links to your micrographs, and the symbolic links could have simpler names with just one variable part containing the micrograph id.

If you'd like to try the current (development) version of Frealix 1.2, I'm happy to share that with you, just let me know.

I should point out I've not played with microtubules for a few years and while in theory Frealix handles microtubule geometries well (even doing the real-space reconstruction trick a la Sindelar), this hasn't been tested or used in a long, long time. In any case, I'm happy to answer questions and update the code as required.

Hope this helps,
Alexis