Faculty Bibliography

Obtaining large, flat, well ordered crystals represents the key to structure determination by electron crystallography. Multilamellar crystals of Ca(2+)-ATPase are a good candidate for this methodology, and we have optimized methods of crystallization and of preparation for cryoelectron microscopy. In particular, high concentrations of glycerol were found to prevent nucleation and to reduce stacking; thus, by seeding solutions containing 40% glycerol, we obtained thin crystals that were 5-30 microns in diameter and 2-10 unit cells thick. We found that removing vesicles and minimizing concentrations of divalent cations were critical to preparing flat crystals in the frozen-hydrated state. Finally, we developed two methods for determining the number of lamellae composing individual crystals, information that is required for structure determination of this crystal form. The first method, using low magnification images of freeze-dried crystals, is more practical in our case. Nevertheless, the alternative method, involving analysis of Laue zones from electron diffraction patterns of slightly tilted crystals, may be of general use in structure determination from thin, three-dimensional crystals

The ATP-driven calcium pump (Ca(2+)-ATPase) is an integral membrane protein (M(r) 110K) which relaxes striated muscle by pumping calcium out of the cytoplasm into the sarcoplasmic reticulum against a large concentration gradient. Recent efforts have attempted to relate the sequence of Ca(2+)-ATPase to its structure and function. In particular, site-directed mutagenesis has identified critical amino-acid residues, and its predicted secondary structure, which includes ten transmembrane helices, has gained experimental support. But direct visualization of the molecule has so far been limited to the cytoplasmic domains at low resolution. We present here the three-dimensional structure of Ca(2+)-ATPase in the native sarcoplasmic reticulum membrane at 14 A resolution, determined by cryo-electron microscopy and helical image analysis. The structure shows an unexpected transmembrane organization, consisting of three distinct segments, one of which is highly inclined. These features can be related to earlier predictions of secondary structure