General Routines

This repository will contain scripts for general data processing of single particle cryo-electron microscopy (cryo-EM) data sets.

Table of contents

• Convert .mrc dm3 files to .mrc (bash script)
• Convert .mrc stack to individual 2D .mrc images
• Extracting particles using makeStack.py
• Estimating CTF using CTFFIND3 and CTFTILT

Convert .mrc dm3 files to .mrc (bash script)

A quick bash script that will convert all micrographs in a given folder that a .dm3 into .mrc format.

./convert_all_dm3_to_mrc.csh

Convert .mrc stack to individual 2D .mrc images

To convert a stack of mrc images into single 2D mrc images. This is helpful for dealing with stacks from from SerialEM or any other stack of .mrc images.

$  General_routines/mrc_stack_to_2Dimages.py
Usage: mrc_stack_to_2Dimages.py -i [stack]

Options:
  -h, --help  show this help message and exit
  -i FILE     Input .mrc stack
  -d          debug

Extracting particles using makeStack.py

###Inputs

makeStack.py assumes that the particles you have picked are stored in a .box file with the same micrograph name as the micrograph, which is in .mrc format. Particle coordinates stored as a .box file are within a text file that has four columns: X, Y, BoxSize X, BoxSize Y.

For instance, the output from [runSignature.py] (https://github.com/leschzinerlab/Signature) will provide particle coordinates in .box format that are compatible with makeStack.py.

####Picking particles

Typically users can pick particles using boxer from EMAN1.9 or e2boxer.py from EMAN2. Both can save particle coordinates in .box format.

####Estimating the CTF

Below, users have the option to extract 1) phase flipped particles or 2) RCT/OTR particle pairs. For either of these processing outputs, you will need to run:

1. phase flipping: CTFFIND3 --> [estimateCTF_CTFFIND3.py] (https://github.com/leschzinerlab/FREALIGN/tree/master/ctffind_ctftilt)

2. RCT/OTR: CTFTILT --> [estimateCTF_CTFTILT.py] (https://github.com/leschzinerlab/FREALIGN/tree/master/ctffind_ctftilt)

These programs will output text files with CTF and tilt information.

###Dependencies

This software requires [SPIDER] (http://spider.wadsworth.org/spider_doc/spider/docs/spider.html) and [EMAN2] (http://blake.bcm.edu/emanwiki/EMAN2) to be installed & libraries correctly setup.

###Running the program

Input options for the program are displayed by running the program without any options on the command line:

$ /General_routines/makeStack.py
Usage: makeStack.py --micros=<micrographs> -o <output stack name.img>

Options:
  -h, --help       show this help message and exit
  --micros=FILE    Path to micrographs with wildcard in quotes ('*en.mrc')
                   (Not needed for tilt mates)
  -o FILE          Output stack name (.img). If tilt mates, do not provide
                   .img extension, output stack name will be the base name for
                   tilted and untilted particles.
  --bin=INT        Binning factor used during boxer picking (Default=1)
  --invert         Optional: Invert contrast of micrographs
  --boxsize=INT    Optional: Box size for final stack. (Default is size used
                   in boxer picking)
  --binstack=INT   Optional: Binning for final extracted particle stack.
                   (Default=1)
  --phaseflip      Flag to phase flip particles
  --ctf=STRING     If phase-flipping - ctf_param.txt output file from
                   estimateCTF_CTFFIND.py if not using tilt mates
  --untilt=STRING  If extracting tilt mates - provide CTFTILT output file for
                   UNtilted micrographs created by estimateCTF_CTFTILT.py.
                   Assumes extension is '01'.
  --tilt=STRING    If extracting tilt mates - provide CTFTILT output file for
                   TILTED micrographs created by estimateCTF_CTFTILT.py.
                   Assumes extension is '00'.
  --noinsideonly   Flag to NOT exclude particles on edges of images (needed
                   for tilt pairs)
  -d               debug

####Extracting raw particles (no phase flipping)

To extract particles from your micrographs without phase flipping or extracting tilt mates for OTR/RCT, you just need to specify the path to the micrographs and provide an output stack name. NOTE The box files are assumed to be the same name as the micrograph names.

$ /General_routines/makeStack.py --micros='*en.mrc' -o outputstack.img

####Extracting particles WITH phase flipping

To extract particles from phase flipped micrographs, you can execute this command, specifying --phaseflip and providing the ctf_param.txt file, which is the output from estimateCTF_CTFFIND.py:

$ /General_routines/makeStack.py --micros='*en.mrc' -o outputstack.img --phaseflip --ctf=ctf_param.txt

####Extracting RCT/OTR tilt mates

work in progress