Tomography - Generating 3D map with Protomo 1.4

save tilt series in both .tif and .dat format and transfer them to Roberts linux computer using SSH file transfer

Move to a directory you put files

tomo-setup.sh

this makes four directories, raw, clean, align, and out. All files are put in to raw directory.

cd raw

move into raw directory

./tomo-clean.sh

notes:if file not present, copy from elsewhere (usually you can find it under /home/roberts-lab/ks/)

it takes while to clean images. .img files are generated.

mv *.img ../clean

format information from save_log.txt into text table

[image name]_001 [Spe dir] [Spe ang] [x middle] [y middle]

example:

series_001 102.26 52.41 1024 1024

series_002 102.26 51.8 1024 1024

...

series_041 102.26 -49.81 1024 1024

series_042 102.26 -51.81 1024 1024

notes:

tab between columns

middle pixel is 1024 for 2048 X 2048 image

easiest to format using OpenOffice spreadsheet

save as [filename].txt in raw/

tomo-create.sh [filename].txt [comment] [filename]-01-itr.tlt

note: single word comment such as "filaments" (w/o quotes) but do not use underbar or hyphen for comment

less [filename]-01-itr.tlt

check format for errors

q to close

mv [filename]-01-itr.tlt ../align

copy a .param file to align/ from elsewhere

rename to [filename]-01.param

note: save at least one *-01.param for future use

nano [filename]-01.param

open a .param file with text editor and modify

imgref = file number of image at 0◦ tilt, e.g.37

check table in raw/[filename].txt if unsure

alismp=3

alibox_x=500 corbox_x=256 refmsk_x=480

alibox_y=500 corbox_y=256 refmsk_y=480

Control+x to exit nano, then press y and return

tomo-ali.sh [filename]-01.param [filename]-01-itr.tlt

A window showing a image pops up

Actions > Align All

Actions > Show Movie, make sure images are aligned properly, if not align them by manually

File > Save

close window

cp [filename]-01.param [filename]-02.param

cp [filename]-01-itr-ali.tlt [filename]-02-itr.tlt

optional step:

tomo-box.sh [filename]-02.param [filename]-02-itr.tlt

tests for errors using specified alignment box

if errors are present

reduce alignment box size until errors are gone

or remove images from .tlt file

tomo-refine.sh [filename]-02.param >[filename]-02.log 2>&1 &

note: to check progress, type:

more [filename]-02.log | grep max

It takes 30 minutes to an hour (depending on for this step, when it is finished

tomo-fit.sh [filename]-02.param

four .ps files showing results of alignment are generated

gimp [filename]-02-fit-cof.ps

§ Import

make sure top curve < 1.01 and bottom curve > 0.99. gimp is image editor like photoshop

cp [filename]-02.param [filename]-03.param

cp [filename]-02-fitted.tlt [filename]-03-itr.tlt

open [filename]-03.param with text editor and modify

alismp=2

alibox_x=750 corbox_x=256 refmsk_x=730

alibox_y=750 corbox_y=256 refmsk_y=730

tomo-refine.sh [filename]-03.param >[filename]-03.log 2>&1 &

It takes a few hours.

tomo-fit.sh [filename]-03.param

gimp [filename]-03-fit-cof.ps

§ Import

make sure top curve < 1.01 and bottom curve > 0.99

cp [filename]-03.param [filename]-04.param

cp [filename]-03-fitted.tlt [filename]-04-itr.tlt

open [filename]-03.param with text editor and modify

alismp=1.5

alibox_x=1000 corbox_x=256 refmsk_x=960

alibox_y=1000 corbox_y=256 refmsk_y=960

tomo-refine.sh [filename]-04.param >[filename]-03.log 2>&1 &

tomo-fit.sh [filename]-04.param

gimp [filename]-04-fit-cof.ps

§ Import

make sure top curve < 1.01 and bottom curve > 0.99

cp [filename]-04.param [filename]-05.param

cp [filename]-04-fitted.tlt [filename]-05-itr.tlt

open [filename]-04.param with text editor and modify

alismp=1.0

alibox_x=1500 corbox_x=256 refmsk_x=1460

alibox_y=1500 corbox_y= 256 refmsk_y=1460

tomo-refine.sh [filename]-05.param >[filename]-05.log 2>&1 &

tomo-fit.sh [filename]-05.param

gimp [filename]-05-fit-cof.ps

§ Import

make sure top curve < 1.01 and bottom curve > 0.99

repeat tomo-refine until two lines in [filename]-xx-fit-cof.ps get close to 1.0

cp [filename]-05.param [filename]-06.param

cp [filename]-05-fitted.tlt [filename]-06-itr.tlt

Optional: put numbers in size of map volume, if you want to specify the volume size

tomo-map.sh [filename]-05.param [filename]-05-itr.tlt

this generate 3D map in mrc format

cd ../out

displayimage [filename]-06-bck.img

check 3D map. Press up and down arrow to go up and down stacks.

Convert 3D map for IMOD:

Open terminal

cd ~/ImageJ

Plugins->MRC reader

Open 3D map

Image->Adjust

Adjust image contrast. Press Apply to apply new setting

Image->Type->8-bit

Convert images to 8-bit

Image->Stacks->Delete Slices

Delete useless slices if there are any

File->Save As->Image Sequence

Save as tif images

move to the directory containing tif files

tif2mrc [filename]-???.tif [filename].mrc

this converts tif files to single mrc file for IMOD

When 3D map is huge use ImageJ on HPC

Open terminal

ssh -x roberts@submit.fsu.edu (or hpcx)

Password required to access to HPC@FSU

cd /ks-scrach/ImageJ

Move to ImageJ directory

./run

Start ImageJ

After adjusting contrast, converting to 8-bit, and saving as tif fles, move a directory containing the tif images

scp *.tif roberts-lab@bio334-pc2.bio.fsu.edu:/home/xxx/xxx/xxx

transfer tif files to Roberts linux

exit

logout HPC

move to the directory containing tif files

tif2mrc [filename]-???.tif [filename].mrc

this converts tif files to single mrc file for IMOD

FOR AMIRA:

sample –cnv –smp 2 2 2 –c –s [map_x/2] [map_y/2] [map_z/2] [filename].thr [filename].bin

preproc –med –m 3 3 3 [filename].bin [filename]-01.med

preproc –med –m 3 3 3 [filename]-01.med [filename]-02.med

preproc –med –m 3 3 3 [filename]-02.med [filename]-03.med

compute [filename].rev = [filename]-03.med / -1

cutimage –fmt mrc [filename].rev [filename]-pretrim.mrc

trimvol [filename]-pretrim.mrc [filename].trim

mrcbyte [filename].trim [filename].mrc