Estimating defocus and astigmatism of micrographs with CTFFIND3

mrcctf.spi

mrcctf.spi takes a micrograph in SPIDER or scanned format, windows it, creates a temporary shell script, and uses the shell script as input to the MRC program ctffind3.exe. This program generates a text report with defocus and astigmatism estimates as well as a power spectrum image. 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 (in SPIDER format)

SPIDER parameter doc file providing

Spherical aberration

Electron beam voltage (kV)

Amplitude contrast ratio

Magnification

Pixel size on scanner (microns)

Selection doc file containing micrograph numbers

Boundary for windowing edges of micrographs

Parameters for ctffind

Box size Box size for ctffind3 (must be even)

ResMin (A)

ResMax (A)

dFMin (A)

dFMax (A)

FStep

Outputs:

Doc file of defocus and astigmatism information

Power spectrum generated by ctffind3

Text report generated by ctffind3

ctffind3

PURPOSE:

Determines defocus and astigmatism for images of arbitrary size. Astigmatic angle is measured from x axis (same conventions as in the MRC 2D image processing programs).

AUTHOR:

CTFFIND3 was developed in 1998 by Nikolaus Grigorieff at the MRC Laboratory of Molecular Biology in Cambridge, The software is licensed under the terms of the GNU Public License version 3 (GPLv3). Download

REFERENCE:

Mindell, JA, Grigorieff N. 2003. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142:334-47.

CTFFIND3 INPUT LINES:

1) Input file name for image

2) Output file name to check result

3) CS[mm], HT[kV], AmpCnst, XMAG, DStep[um]

4) Box, ResMin[A], ResMax[A], dFMin[A], dFMax[A], FStep

CTFFIND3 PARAMETERS:

CS: Spherical aberration coefficient of the objective in mm

HT: Electron beam voltage in kV

AmpCnst: Amount of amplitude contrast (fraction). For ice images 0.07, for negative stain about 0.15.

XMAG: Magnification of original image

DStep: Pixel size on scanner in microns

Box: Tile size. The program divides image into square tiles and calculates the average power spectrum. Tiles with a significatly higher or lower variance are excluded; these are parts of the image which are unlikely to contain useful information (beam edge, film number etc). IMPORTANT: Box must have even pixel dimensions.

ResMin: Low resolution end of data to be fitted.

ResMaX: High resolution end of data to be fitted.

dFMin: Starting defocus value for grid search in Angstroms. Positive values represent an underfocus. The program performs a systematic grid search of defocus values and astigmatism before fitting a CTF to maching precision.

dFMax: End defocus value for grid search in Angstroms.

FStep: Step width for grid search in Angstroms.

CTFFIND3 OUTPUTS:

1) Power spectrum generated (in SPIDER format).

This output image file can be used to check the result of the fitting. It shows the filtered average power spectrum of the input image in one half, and the fitted CTF (squared) in the other half. The two halfs should agree very well for a sucessfull fit. Example output.

2) Text report.

readmrc.py

PURPOSE:

Searches through the CTFFIND3 report for the following lines:

DFMID1	DFMID2	ANGAST	CC1
19240.91	18631.46	6.43	0.0137	Final Values

Where:

DFMID1 = defocus along "long" axis

DFMID2 = defocus along "short" axis

ANGAST = angle of astigmatism

CC = magnitude of astigmatism

OUTPUT:

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

key	n	mic no	avg defocus	defocus1	defocus2	angle	astig
1	6	1.000	26404.675	26446.72	26362.63	0.940	0.013

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

NOTES:

Angle and magnitude of astigmatism are represented differently than in SPIDER. According to Grigorieff's web site:

Question: Is there a program to CTF correct a single image using the values obtained from ctffind3 (i.e. correction taking into account some astigmatism)?

Answer: SPIDER can do this -- you would use the "TF C" function to generate CTF with amplitudes, or "TF CT" to just generate the binary version for phase flipping. Then you would multiply the FT of your image by the result using "MU". The only tricky thing is converting the CTFFIND3 parameters to SPIDER ones, because the astigmatism angle conventions are different. The following code illustrates:

spider_defocus = (DFMID1 + DFMID2)/2;

spider_astig = (DFMID2 - DFMID1);

spider_angle_astig = ANGAST - 45;

if (spider_astig < 0)

   {spider_astig = -spider_astig;

   spider_angle_astig = spider_angle_astig + 90;}

enhancepowmrc.spi

enhancepowmrc.spi enhances the power spectrum image from CTFFIND3.

Inputs:

Power spectra generated by SPIDER

Power spectra generated by CTFFIND3

Selection doc file containing micrograph numbers

Source: defmrc.html     Page updated: 9/22/10     Author: Bill Baxter