Appion » TiltPicker

A copy of this page is available at:

http://emg.nysbc.org/projects/tiltpicker/wiki/TiltPicker_Usage

BUG TRACKING:
----------------------¶

TiltPicker bugs are now tracked via the AMI redmine server, visit
 http://emg.nysbc.org/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 from the appion.org website:

http://code.google.com/p/appion/downloads/list

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