Manual tilt pair particle picking - cianfrocco-lab/Old-school-processing GitHub Wiki

Home > Manual tilt pair particle picking

You'll use e2RCTboxer.py (part of the EMAN2 package) to pick tilt mates. It simply takes the untilted and tilted images as inputs, along with the box size desired for picking:

e2RCTboxer.py untilted.mrc tilted.mrc --boxsize=128

This command will open four windows: a window for each micrograph, a menu, and a display window for picked particles.

1. To start, make sure that the Current Boxing Tool is set to Pair Picking - this will allow 'semi-automated' picking of the tilt pairs, where picks that you select on the untilted images are automatically transferred to the tilted image. Also, make sure Update box positions and Center opposite box position are selected.

2. Find a particle that it unambiguous on both untilted and tilted micrographs. Click on the particle in the untilted image (boxes will be blue and numbered). Now click on the particle in the tilted image. They should each have the same number.

3. After picking the first 3 particles, the program will begin to try to 'predict' where the tilted particle actually is in the tilted image after you select a particle in the untilted image. If it is wrong, you can adjust the particle in the tilted image by clicking and dragging the box around the particle.

4. The program will eventually get quite good at matching the two images, allowing you to just focus on manual particle picking in the untilted image. You'll see that the program has a lag time between when you 'click' on the untilted image and when the actual boxes appear. You can still speed along picking even if it is slow; the program will catch up to whatever number of points that you click.

5. When you are finished, click Write Boxes in the menu window to save the particle coordinates as .box files. The program will automatically name the .box file after the .mrc micrograph input.