Focus Documentation

By Vinzenz Unger and Anchi Cheng

The following section provides a general description how to analyze (low-dose) electron micrographs of two-dimensional crystals. In summary the whole procedure can be divided into 5 steps:

• Step 1:Optical diffraction, image acquisition and preparation, determination of lattice parameters, calculation of unit cell dimensions and magnification, calculation of a filtered image.
• Step 2: Correction of lattice distortions, extraction of raw image data and correction of phases for the effect of the contrast transfer function (CTF).
• Step 3: Determination of plane group symmetry.
• Step 4:Combination of data from different images, data refinement, data averaging and generation of projection density maps.
• Step 5:Determination of tilt geometries.
• Step 6:Merging of 3D data, lattice line fitting, data refinement and calculation of 3D density maps.

A number of examples are given to explain the effect of certain procedures and to illustrate the impact of inaccurately determined image parameter. All examples are taken from a study investigating the structure of a recombinant gap junction channel. The larger thickness (>150Å) of this specimen causes some of the illustrated effects to be very pronounced. If a thin specimen (~ 80Å thick) is analyzed some of these effects are more subtle yet it seemed more reasonable to give examples that are easy to understand.

Following this general account is a more detailed description of the current MRC program package that also provides some more in depth information about how to optimize the processing steps. Finally, a number of explicit protocols can be found at the end of this chapter. The protocols cover only the basic routines due to space limitations.