Defocus Estimation with MRC CTFFIND2

Estimating defocus and astigmatism with MRC CTFFIND2

The following procedures estimate defocus and astigmatism from micrographs.

mrc.spi The main SPIDER procedure file. It calls two scripts:

ctffind, a shell script, that calls ctffind2.exe, and

readmrc.py, a python script that converts the report generated by ctffind2 into a SPIDER document file.
** Note to Albany users: you do not have to download this script, it should already be on your path.

Both scripts must be executable (e.g., run the unix command chmod 775 ctffind after downloading that file).

mrc.spi

mrc.spi takes a micrograph in SPIDER format, converts it to MRC format, then sends it as input to a shell script (ctffind) that calls the MRC program ctffind2.exe. This program generates a text report with defocus and astigmatism estimates as well as a power spectrum image. The latter is converted to SPIDER format by the procedure file. It then calls a python script ( readmrc.py), which searches the report for the defocus and astigmatism values, and writes these out to a SPIDER document file.

Inputs:
    micrographs
    start, end micrograph numbers
    parameters to ctffind, including
        - spherical aberration
        - electron beam voltage (in kV)
        - amplitude contrast ratio
        - magnification
        - pixel size on scanner (in microns)

Outputs:
    doc file of defocus and astigmatism information.
    power spectrum generated by ctffind2.
    text report generated by ctffind2.

ctffind

This shell script makes the actual call to ctffind2.exe. Inside the script, the program name is followed by 4 lines of parameters:

ctffind2
input_micrograph (in mrc format)
output_power_spectrum (in mrc format)
CS[mm], HT[kV], AmpCnst, XMAG, DStep[um]
Box, ResMin[A], ResMax[A], dFMin[A], dFMax[A], FStep

The first 3 parameter lines are handled by the calling procedure file. The last line (Box, ResMin[A], ResMax[A], dFMin[A], dFMax[A], FStep) has default values in the ctffind script which users may want to change. View the script for more information.

While running, ctffind2 prints out a report of its ongoing parameter search. mrc.spi redirects this information into a text file. An example report generated by ctffind2.

ctffind does generate a power spectrum image with the estimated model on the left and data on the right. However, because of problems with dynamic range, it is very difficult to see the data in Web. The procedure file fixmrc.spi creates an image with the MRC-generated model on the left, and the SPIDER-generated power spectrum on the right. Example output

readmrc.py

This script searches through the report for the lines

      DFMID1      DFMID2      ANGAST          CC

    19240.91    18631.46        6.43     0.01375  Final Values

where
DFMID1 = defocus along "long" axis
DFMID2 = defocus along "short" axis
ANGAST = angle of astigmatism
CC = magnitude of astigmatism

The two defocus measures are averaged, and this information is written out to a SPIDER document file with 6 columns:

  key      mic no.  avg defocus  defocus1    defocus2   angle   astig

    1 6     1.000   26404.675   26446.720   26362.630   0.940   0.013

The the micrograph number is used as the index key. If there are gaps in the micrograph numbers, use the SPIDER command DOC REN on the file.

NB: Angle and magnitude of astigmatism are represented differently than in SPIDER.

Back to Single Particle Reconstruction

Source: defmrc.html Created: 1/9/03 Bill Baxter