2DHybrid Analysis.

We have developed an approach termed '2D hybrid analysis' for building three-dimensional (3D) structures from electron microscopy (EM) images of biological molecules. The key advantage is that it is applicable to flexible molecules, which are difficult to analyze by the approach in which 3DEM maps are reconstructed. In the proposed approach, a large number of atomic models with different conformations are first built by computer simulation. Then, simulated EM images are produced from each atomic model. Finally, these images are compared with an experimental EM image to identify the best-fitting atomic model. Two kinds of models are used to simulate the EM images: the negative-stain model and the simple projection model. Although the former is more realistic, the latter permits faster computation. We applied this approach to the averaged EM images of integrin. Although many of these were reproduced well by the best-fitting atomic models, others did not closely resemble any of the simulated EM images. However, the latter group were well reproduced by averaging multiple simulated EM images originating from atomic models with rather different conformations or orientations. This indicated that our approach is capable of detecting mixtures of conformations in the averaged EM images, which should assist in their correct interpretation.