Appion » TiltPicker

A copy of this page is available at:

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

h2. BUG TRACKING:

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 TiltPicker bugs are now tracked via the AMI redmine server, visit

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

 to file any bugs

h2. YOUTUBE MOVIE:

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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/

h2. LAUNCHING THE PROGRAM:

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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.

h2. STEP BY STEP TUTORIAL:

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* 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	

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* 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

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* 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

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* Transfer pick particles

** Pick a single particle in only one image.

** Use transfer or *Xfer picks* button -- this automatically transfers the pick to the 

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* 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

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* 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

h2. BUTTON DOCUMENTATION:

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Notes: 

# 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.

# If the button name is followed by three dots '...', it means a pop up window will be displayed for more input.

# 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