Spherical reconstruction: A method for structure determination of membraneproteins from cryo-EM images

We propose a new method for single-particle reconstruction, which should be generally applicable to structure determination for membrane proteins. Aft er reconstitution into a small spherical vesicle, a membrane protein takes a particular orientation relative to the membrane normal, and its position in the projected image of the vesicle directly defines two of its three Eul er angles of orientation. The spherical constraint imposed by the vesicle e ffectively reduces the dimensionality of the alignment search from 5 to 3 a nd simplifies the detection of the particle. Projection images of particles in vesicles collectively take all possible orientations and therefore cove r the whole Fourier space. Analysis of images of vesicles in ice showed tha t the vesicle density is well described by a simple model for membrane elec tron scattering density. In fitting this model we found that osmotically sw ollen vesicles remain nearly spherical through the freezing process. These results satisfy the basic experimental requirements for spherical reconstru ction. A computer simulation of particles in vesicles showed that this meth od provides good estimates of the two Euler angles and thus may improve sin gle-particle reconstruction and extend it to smaller membrane proteins. (C) 2001 Academic Press.