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Usage

A copy of this page is available at: TiltPicker Usage

BUG TRACKING

TiltPicker bugs are now tracked via the AMI redmine server, visit http://emg.nysbc.org/redmine/projects/tiltpicker/ to file any bugs

YOUTUBE MOVIE

I just made a screen capture video on how to use it. It is available is low resolution on youtube:

http://www.youtube.com/watch?v=cw1osPBY_JM

or you can download the full screen version.

I recommend using VLC to watch it:

http://www.videolan.org/vlc/

LAUNCHING THE PROGRAM

To run the program, type: 

$ ./ApTiltPicker.py --1 image1.mrc --2 image2.mrc -p tiltpicks.spi
# 'tiltpicks.spi' is optional. I left an example in the tiltpicker folder. You must run the program in the tiltpicker folder. To run the example type:
$ ./runPicker.sh

If all works the picker will open up and you'll have two images of ribosomes.

STEP BY STEP TUTORIAL

  • Start TiltPicker
    • Extract TiltPicker, on command line type:

tar -zxf tiltpicker-2.0.tgz

Go to tiltpicker directory, on command line type:

cd tiltpicker

Display TiltPicker help, on command line type:

./ApTiltPicker.py -h

Open TiltPicker, on command line type:

./ApTiltPicker.py -l data/rawu048b.jpg -r data/rawu049b.jpg -t 50.0

  • Initialize picking:
    • Maximize window
    • Set correct zoom for images, if necessary
    • Center the middle bar, if necessary
    • Manually pick 5 partcle pairs from each image
    • Save picks to a file, file will then auto-save all subsequent picks
  • Initialize alignment:
    • Hit the Apply button, circles will NOT match crosses
    • Find theta (tilt angle)
      • Hit Theta... button
      • Hit Run
      • This number should match the value from Goniometer
      • Hit Apply to accept this value
    • Optimize inplane rotation angles and shift
      • Refine means this parameter will be optimized
      • Lock means this parameter is locked and will NOT be optimized
      • Hit Optimize...
      • Hit Run a few times
      • Hit Apply
    • Use Mask button to only look at overlapping regions, overlap % is shown on status bar
    • Hit Auto Optimize, it automatically performs theta, optimize, and mask in a single click
    • If you look at the Terminal, it will tell you how many optimization rounds were performed
  • Transfer pick particles
    • Pick a single particle in only one image.
    • Use transfer or Xfer picks button --- this automatically transfers the pick to the
  • DoG pick particles
    • Measure particle size using ruler tool (at top), Use 1X zoom in to get accurate value
    • Sometimes it is best to use a 10% lower estimate for the diameter, e.g., use 30 if you measure 34
    • You can run DoG picker with wrong contrast and reject picks
    • Run DoG picker with correct contrast
    • Show terminal
    • Accept picks
    • If not enough picks were made lower the threshold
    • Look at DoG map images
    • If the peaks are not crisp dot and more like fuzzy balls, try reducing the particle diameter
  • Clean up particle picks
    • Use clear bad picks button
    • Use polygon tool
    • Auto optimize
    • Use cutoff button to remove picks below a threshold
    • Add more picks
    • Save and quit

BUTTON DOCUMENTATION

Notes:

  1. The underlined letter on the button means that you can use the Alt button (Option on a Mac) and that letter to push the button.
  2. If the button name is followed by three dots '...', it means a pop up window will be displayed for more input.
  3. All buttons are also availale at the top menu

(from left to right)

  • Apply (Alt-A) --- Displays picked particle locations from one image in the other image as circles

  • Theta... (Alt-T) --- Determine the tilt angle difference between images independent of tilt axis rotation (positive, untilted on left; negative, untilted on right)

  • Optimize... (Alt-O) --- Perform least squares refinement of all alignment parameters
  • Mask (Alt-M) --- Hide non-overlapping regions in the images (based on alignment parameters)

  • Auto Optimize (Alt-Z) --- Automatically perform above 3 steps: Theta, Optimize, and Mask in single click

  • Rm Worst Picks (Alt-W) --- Remove the worst picks by alignment error highlighted by a yellow plus (+) symbol

  • Cutoff... (Alt-C) --- Remove the worst picks by specifying an alignment error
  • Xfer picks (Alt-X) --- Transfer picks from one image to the other based on the alignment parameters
  • Rm Polygon (Alt-P) --- Remove all points located within a drawn polygon from both images, good for large pieces of crud

  • Reset... --- Reset all parameters and remove all picks, i.e., start over.

  • Dog Pick... (Alt-D) --- Run automated particle picker. Requires an estimate of the size of the particle (in pixels)

  • Quit (Alt-Q) --- Quit the program