Three-dimensional crystals of Ca2+-ATPase from sarcoplasmic reticulum: Merging electron diffraction tilt series and imaging the (h,k,0) projection

Electron crystallography offers an increasingly viable alternative to X-ray crystallography for structure determination, especially for membrane prote ins. The methodology has been developed and successfully applied to 2D crys tals; however, well-ordered thin, 3D crystals are often produced during cry stallization trials and generally discarded due to complexities in structur e analysis. To cope with these complexities, we have developed a general me thod for determining unit cell geometry and for merging electron diffractio n data from tilt series. We have applied this method to thin, monoclinic cr ystals of Ca2+-ATPase from sarcoplasmic reticulum, thus characterizing the unit cell and generating a 3D set of electron diffraction amplitudes to 8 A ngstrom resolution with tilt angles up to 30 degrees. The indexing of data from the tilt series has been verified by an analysis of Laue zones near th e (h, k, 0) projection and the unit cell geometry is consistent with low-an gle X-ray scattering from these crystals. Based on this unit cell geometry, we have systematically tilted crystals to record images of the (h, k, 0) p rojection After averaging the corresponding phases to 8 Angstrom resolution , an (h, k, 0) projection may has been calculated by combining image phases with electron diffraction amplitudes. This map contains discrete densities that most likely correspond to Ca2+-ATPase dimers, unlike previous maps of untilted crystals in which molecules from successive layers are not aligne d. Comparison with a projection structure from tubular crystals reveals dif ferences that are likely due to the conformational change accompanying calc ium binding to Ca2+-ATPase. (C) 1998 Academic Press.