Preparation and analysis of large, flat crystals of Ca(2+)-ATPase for electron crystallography

Abstract

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.

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

“…Thus, Laue zones are most prominent near the a*-b* plane (h, k, 0 projection), or possibly near other principal projections. In addition, crystal thickness can be judged by the width of the Laue zones (Shi et al, 1995), which re¯ects the width of the sinc functions ( Figure 1c). …”

“…In previous studies of thin, 3D crystals, both by ourselves (Shi et al, 1995) and by others (Leapman et al, 1993;Rez et al, 1992), much effort has been made to account for the thickness of crystals. This is because the sampling of Fourier data along c* is very much dependent on this thickness, especially with a small number of unit cells along this axis.…”

“…Alternatively, crystals were nucleated at low temperature, brought to the higher temperature for $seven days and then returned to the lower temperature, or simply left at low temperature for their entire nucleation and growth period. These different temperature regimens were designed to produce the largest, thinnest, most uniform crystals possible (Shi et al, 1995).…”

“…Ca 2 -ATPase from skeletal sarcoplasmic reticulum offers a good example of thin, 3D crystals (Taylor et al, 1988) and produces electron diffraction to $3.5 A Ê resolution (Shi et al, 1995). The requirement for saturating concentrations of calcium suggest that Ca 2 -ATPase adopts the conformation known as E 1 Á Ca 2 in these crystals (Stokes & Lacapere, 1994), in which calcium binding to transport sites has activated the nucleotide site for phosphorylation by ATP (MacLennan et al, 1997).…”

“…The individual lamellae consist of a mixed bilayer of lipid and detergent with Ca 2 -ATPase molecules protruding from both sides of this bilayer ( Figure 1a). Lamellar stacking is mediated by the cytoplasmic domains of Ca 2 -ATPase and attempts to produce 2D crystals have failed (Misra et al, 1991;Shi et al, 1995); in fact, these attempts provide circumstantial evidence that stacking is essential to crystal nucleation. Symmetry and unit cell dimensions within the lamellar plane have been documented in images and in electron diffraction patterns from thin, plate-like crystals (a 166.4 A Ê and b 55.7 A Ê ), whereas small, cylindrical crystals grown under slightly different conditions have been used to measure unit cell dimensions normal to the lamellae (Cheong et al, 1996;Ogawa et al, 1998).…”