Spherical reconstruction: a method for structure determination of membrane proteins from cryo-EM images.
We propose a new method for single-particle reconstruction, which should be generally applicable to structure determination for membrane proteins. After 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 Euler angles of orientation. The spherical constraint imposed by the vesicle effectively reduces the dimensionality of the alignment search from 5 to 3 and simplifies the detection of the particle. Projection images of particles in vesicles collectively take all possible orientations and therefore cover the whole Fourier space. Analysis of images of vesicles in ice showed that the vesicle density is well described by a simple model for membrane electron scattering density. In fitting this model we found that osmotically swollen vesicles remain nearly spherical through the freezing process. These results satisfy the basic experimental requirements for spherical reconstruction. A computer simulation of particles in vesicles showed that this method provides good estimates of the two Euler angles and thus may improve single-particle reconstruction and extend it to smaller membrane proteins.
Jiang
Q X
QX
Department of Cellular and Molecular Physiology, Yale University, 333 Cedar Street, New Haven, Connecticut 06520, USA.
Chester
D W
DW
Sigworth
F J
FJ
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry; D003198:Computer Simulation; D020285:Cryoelectron Microscopy / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D021621:Imaging, Three-Dimensional / Q000379:methods; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry*; D010316:Particle Size; D010743:Phospholipids / Q000737:chemistry; D011487:Protein Conformation
D008081:Liposomes; D008565:Membrane Proteins; D010743:Phospholipids; D000252:Calcium-Transporting ATPases