Tutorials on Random Conical Tilt

Random conical tilt is a method for determining 3-D structure using tilt-pairs. Typically this is done with an untilted image and a second image tilted to 45-60 degrees. Unfortunately, this strategy leads to structures which always have a 'missing cone'. An alternative strategy is the +/- 45 degree tilt method, where images are collected at -45 degrees and +45 degrees. Since they are then 90 degrees apart, it is possible to do a reconstruction without a missing cone, provided some other conditions are met. More specifically, the particles being imaged must be in a nearly random orientation in the ice. The original RCT method took advantage of the fairly common preferred particle orientation problem in negative stain, but this results in a final structure which always has a missing cone. If particles are randomly oriented, however, then by averaging together multiple RCT reconstructions, the missing wedge can be filled in.

This technique generally does not provide very high quality structures, but can be very useful when the structure of your assembly is completely unknown, as the tilted image gives additional information about the 3-D orientation of the particles, not available to traditional single particle reconstruction methods. It is also often used with very small molecules, in negative stain, where traditional single particle reconstruction is difficult.

There are two tutorials. One relies on a real data set which has not yet been released. The other uses simulated data (provided).

• Tutorial on using EMAN2 for Random Conical Tilt reconstructions

  • (data not yet released)

• Tutorial on using EMAN2 for Random Conical Tilt reconstructions using simulated data

  • Simulated RCT Data