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Filament selection

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charleswain
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Joined: 12 Apr 2017

Questions about the filament selection and the saved box files.

In the tutorial (Ab(40) using Frealix) on line (http://grigoriefflab.janelia.org/frealix/abeta_tutorial), it said:

"With "Select" selected on the tools window, begin by picking an endpoint. This endpoint (as shown by the green arrow) should be located along the filament near its starting end, and should not be a crossover.
Following the filament, select the next crossover points in order.
After the last crossover has been selected, pick an endpoint near the end of the filament."
1. This description gives my first set of questions, does this mean the first box and the last box should not be the crossover? Or should I just select the crossovers with boxes.

"When a full filament has been selected, click on Boxes -> Save Box DB in the tool box (or Ctrl+S) to save your box file in the "boxes" directory.
After the file has been saved, click on Boxes -> Clear Boxes in the tool box.
Repeat the previous steps until all good filaments in the films have been picked."
2. Does this mean, I should save a box file for each filament? If I have 10 filaments in each image, I should have 10 box files for each image, which means I will have about 1000 box files since I have about 100 images. I was told the name of box file should match with the mrc file name. Do you have any suggestions about the name of the box files? Also the data size will be extremely large if I have 1000 box files which will bring me more difficulties. Any suggestions in terms of the data size?

Thanks!

Alexis
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Joined: 5 May 2009
Hello, Thanks for trying

Hello,

Thanks for trying Frealix.

does this mean the first box and the last box should not be the crossover?

Yes, in the case of abeta fibrils, I have found that it is helpful to pick an extra point before the first crossover and an extra point after the last crossover ("first-and-last waypoints", abbreviated to "FAL_WPS" in some options). Later on, the parts of the filament between the first waypoint and the first crossover, and between the last waypoint and the last crossover, can be ignored during 3D reconstruction or even by the scoring function.

Does this mean, I should save a box file for each filament? If I have 10 filaments in each image, I should have 10 box files for each image, which means I will have about 1000 box files since I have about 100 images.

Yes, this is correct. It is very inconvenient. This is why there is an alternative way to pick filaments (see below).

New way to pick filaments

I modified the display program from Tigris so that it can read & write Frealix waypoints.ali files natively. This is convenient because it allows picking of many filaments much more easily (though it is still a manual process).

To give you an idea of how I have used it in the past:

  1. Create an MRC stack of all your micrographs. You may want to bin/resample the micrographs so that the file isn't too large.
  2. Open the stack with Tigris Display
  3. Within Display, switch to Waypoint Selection mode (Select > Waypoint Selection Mode)
  4. Start clicking on waypoints (left click; waypoints can be deleted by Ctrl-click or moved by click-drag)
  5. When done clicking for a filament, click the "+" button next to the filament number. This starts a new filament. To go back to a previous filament, use the "-" button
  6. When done clicking on filaments for this micrograph, go to the next micrograph by clicking the "+" button on the left-hand-side of the toolbar

I hope this is enough information to help you get started - it should save you a lot of time for picking & it's also very useful for inspecting results from refinement. Don't hesitate to ask more questions as needed.

Cheers,
Alexis

charleswain
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Joined: 12 Apr 2017
May I get your personal

May I get your personal email/cell phone? My email is bing.wangATnih.gov. I will definitely ask more questions about the frealix. It could be more efficient. I really want to get this part of job done quickly. We could post the question/answers after that. I think it will help us to use/learn/expand/distribute this good software too. :)

Thank you