Finding Defocus with Ctfplotter for Three Example Tilt Series

(IMOD 4.9)

University of Colorado, Boulder

 

This document will guide you through running Ctfplotter to find defocus for three different tilt series, one where the signal of the CTF is very strong, one where it is weak, and one where it is sometimes strong and sometimes weaker.  It will explain some of the most important aspects of the process.  For more details, consult the Guide to Ctfplotter, which fully explains each aspect of the interface and also has screen shots based on these two data sets.  Labels in the Etomo or 3dmod interface are shown in Bold, and entries in fields are shown in italics.

 

Determining Defocus in a Tilt Series from a K2 Camera:

 

This tilt series, from Cindi Schwartz, is of a flagellum of a Giardia cell, taken with a K2 camera on a Krios microscope at Janelia Farm.  The total dose was 26 electrons/square Angstrom.  Images were taken in superresolution mode with an exposure time of 0.5 sec to avoid having to save and align subframes, and reduced by a factor of 4 with antialiasing.  With this protocol, they may have somewhat better high-frequency information than a typical tilt series taken in counting mode without binning, so the power spectra may be particularly good here.

 

Determining Defocus in a Tilt Series from a CCD camera:

 

This tilt series is of a preparation of bovine papilloma virus (BPV), taken by Mary Morphew on an F20 with a US4000 CCD camera at a nominal defocus of -3 microns.  It is the series used in the Ctfplotter Guide to illustrate program operations.  Here, it illustrates some of the challenges in doing CTF correction on relatively low-defocus tilt series take with a CCD, which provides poorer information at high frequencies than a direct detector.

 

Determining Defocus in a Tilt Series from a DE-12 Camera:

 

This tilt series, from Cindi Schwartz, is of a preparation of microtubules decorated with the motor protein Eg5, taken with a DE-12 camera during a demo on the F20 microscope in Boulder.  The tilt series had a 2 degree increment and the total dose was 79 electrons/square Angstrom.  Parts of the series have good signal for determining CTF, but not all of it.