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 proteins. The methodology has been developed and successfully applied to 2D crystals; however, well-ordered thin, 3D crystals are often produced during crystallization trials and generally discarded due to complexities in structure analysis. To cope with these complexities, we have developed a general method for determining unit cell geometry and for merging electron diffraction data from tilt series. We have applied this method to thin, monoclinic crystals of Ca2+-ATPase from sarcoplasmic reticulum, thus characterizing the unit cell and generating a 3D set of electron diffraction amplitudes to 8 A 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 the (h, k, 0) projection and the unit cell geometry is consistent with low-angle 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) projection. After averaging the corresponding phases to 8 A resolution, an (h, k, 0) projection map 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 aligned. Comparison with a projection structure from tubular crystals reveals differences that are likely due to the conformational change accompanying calcium binding to Ca2+-ATPase.
Shi
D
D
Department of Cell Biology, NYU School of Medicine, 540 First Avenue, New York, NY, 10016, USA.
Lewis
M R
MR
Young
H S
HS
Stokes
D L
DL
D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D003461:Crystallography / Q000379:methods*; D004583:Electrons; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014961:X-Ray Diffraction
D000252:Calcium-Transporting ATPases