Picking particles with Tiltpicker

Before beginning, it is important to note that Ed Brignole's scripts require a specific nomenclature and micrograph order.

Tilted micrographs must be odd numbered, and before the untilted micrograph.

(This is not strictly true, but it is important that when the micrograph list is generated, this be true).

Second, picked particles must be numbered according to the odd-numbered, tilted micrographs. (e.g pik0001.spi go with micrograph micro0001, pik0003 goes with micrograph micro0003).

Tilted micrographs will thus occupy positions 1,3,5, etc...., and picked particles will be numbered the same.

In order to pick the particles themselves, I have generated a short script, which extracts the micrograph pair from an mrc stack using imagic, and starts up Tiltpicker.

copy to script.csh or .com, and run. (some modification of the paths may be required).

This script currently uses a micrograph pair mentality (e.g. running the command "./script.com 1" will display micrographs 1 and 2, running "./script.com 2" will display micrographs 3 and 4).

Finally, if you wish to use the automatic particle picker, pick 3 to 4 particles in each corner of the image, run "Find Theta" and "Optimize Angles"

Then click on Run Dogpicker, enter the diameter of your particle (you can measure this using the ruler tool), I don't change the threshold, but you can experiment with doing this.

Change the number of peaks to around 50% more than the number of particles you expect (I use 150 for fields having around 100 decent particles).

Finally, carefully check the micrographs for "bad" particles (wrong size, aggregated, etc...).

It is VERY important to delete the particles in pairs, and well. Otherwise you will throw off the angular determination, which you should run until it is stable before saving the particles.

If you find the angle is badly off, zooming out to 1/3 or 1/6 so you can see the whole micropgraph will sometimes allow you to pinpoint which particle you don't have a pair mate for. This will usually solve the problem. Using the automatic fixing functions is a BAD idea, as this seldom works.

Remember, any on screen click registers as a particle, even if it ends up outside of the micrograph, so it is sometimes a good idea to zoom very far out, if there is no obvious culprit.

Good luck.

script1.com

#!/bin/csh -f
cd path/to/stack/

setenv IMAGIC_BATCH 1

if ($#argv < 1) then
    echo "enter the tilt pair wanted, you fool"
    exit
endif
@ x1 = ($1 * 2)
echo $x1
@ x2 = $x1 + 1
echo "! "
echo "! "
echo "! ====================== "
echo "! IMAGIC ACCUMULATE FILE "
echo "! ====================== "
echo "! "
echo "! "
echo "! IMAGIC program: excopy -----------------------------------------------"
echo "! "
/gusr/imagic/linux/imagic/incore/excopy.e <<EOF
2
EXTR
101024_ccomplex
temp1
INTERACTIVE
$x1
EOF


/gusr/imagic/linux/imagic/incore/excopy.e <<EOF
2
EXTR
101024_ccomplex
temp2
INTERACTIVE
$x2
EOF



# convert temp1 and temp2 to mrc to be read into tiltpicker

echo "! "
echo "! IMAGIC program: em2em ------------------------------------------------"
echo "! "
/gusr/imagic/linux/imagic/stand/em2em.e <<EOF
IM
MRC
2D
SINGLE_2D
temp1
temp1_$1
0
mrc
NO
EOF
echo "! "
echo "! IMAGIC program: em2em ------------------------------------------------"
echo "! "
/gusr/imagic/linux/imagic/stand/em2em.e <<EOF
IM
MRC
2D
SINGLE_2D
temp2
temp2_$1
0
mrc
NO
EOF

cd ../doc/

# set up environment for python
switch (`uname -m`)
  case x86_64
    setenv PYTHONPATH /gware/python/lib64/python2.4/site-packages:/gware/python/lib64/python2.4/site-packages/PIL:/gware/python/lib64/python2.4/site-packages/wx-2.8-gtk2-unicode
    setenv LD_LIBRARY_PATH /gware/atlas3.8.3/linux64/lib:/gware/wxPython-2.8.9.2/linux64/lib
  breaksw

  default:
    setenv PYTHONPATH /gware/python/lib/python2.4/site-packages:/gware/python/lib/python2.4/site-packages/PIL:/gware/python/lib/python2.4/site-packages/wx-2.8-gtk2-unicode
    setenv LD_LIBRARY_PATH /gware/atlas3.8.3/linux/lib:/gware/wxPython-2.8.9.2/linux/lib
  breaksw
endsw


/gware/tiltpicker/ApTiltPicker.py -1 ../raw/temp1_$1.mrc -2 ../raw/temp2_$1.mrc

rm ../images/temp*