Dealing with particles in different conformational states by electron microscopy and image processing

Electron microscopy and image processing are powerful tools for investigating different conformational states of enzymes. It is not always possible to isolate these often unstable intermediates as single species. As a result electron micrographs show a snapshot of enzymes in various conformational states. We describe here how to recognize that the imaged particles have different conformations and how to obtain for each species a three-dimensional model using single-particle image processing. We investigated the ATP synthase from chloroplasts, which has a molecular mass of about 550 kDa. It is a membrane-bound enzyme and consists of two segments, a membrane-embedded hydrophobic F(0) part and a hydrophilic F(1) part. Analysis of the particle images indicated that the molecules were in two different conformations. For both conformations three-dimensional models were calculated, which showed that the structures differed mainly in the tilt of the F(0) part with respect to the F(1) part.