Preparation and Analysis of Large, Flat Crystals of Ca(2+)-ATPase for Electron Crystallography

Overview

Journal Biophys J

Publisher Cell Press

Specialty Biophysics

Date 1995 Mar 1

PMID 7756535

Citations 7

Authors

D Shi

H H Hsiung

R C Pace

D L Stokes

Affiliations

Soon will be listed here.

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.

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Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump.

Glaves J, Trieber C, Ceholski D, Stokes D, Young H J Mol Biol. 2010; 405(3):707-23.

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Young H, Stokes D J Membr Biol. 2004; 198(2):55-63.

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Two-dimensional crystallization of Ca-ATPase by detergent removal.

Lacapere J, Stokes D, Olofsson A, RIGAUD J Biophys J. 1998; 75(3):1319-29.

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References

Lamvik M, Davilla S . Calibration methods for quantitative image processing in electron microscopy. J Electron Microsc Tech. 1989; 11(2):97-101. DOI: 10.1002/jemt.1060110202. View

Taylor K, Mullner N, Pikula S, Dux L, Peracchia C, Varga S . Electron microscope observations on Ca2+-ATPase microcrystals in detergent-solubilized sarcoplasmic reticulum. J Biol Chem. 1988; 263(11):5287-94. View

Stokes D, Green N . Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing. Biophys J. 1990; 57(1):1-14. PMC: 1280637. DOI: 10.1016/S0006-3495(90)82501-7. View

Stokes D, Green N . Structure of CaATPase: electron microscopy of frozen-hydrated crystals at 6 A resolution in projection. J Mol Biol. 1990; 213(3):529-38. DOI: 10.1016/s0022-2836(05)80213-x. View

Henderson R, Baldwin J, Ceska T, Zemlin F, Beckmann E, Downing K . Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol. 1990; 213(4):899-929. DOI: 10.1016/S0022-2836(05)80271-2. View

Wang D, Kuhlbrandt W . High-resolution electron crystallography of light-harvesting chlorophyll a/b-protein complex in three different media. J Mol Biol. 1991; 217(4):691-9. DOI: 10.1016/0022-2836(91)90526-c. View

Jap B, Walian P, Gehring K . Structural architecture of an outer membrane channel as determined by electron crystallography. Nature. 1991; 350(6314):167-70. DOI: 10.1038/350167a0. View

Misra M, Taylor D, Oliver T, Taylor K . Effect of organic anions on the crystallization of the Ca2(+)-ATPase of muscle sarcoplasmic reticulum. Biochim Biophys Acta. 1991; 1077(1):107-18. DOI: 10.1016/0167-4838(91)90532-5. View

Glaeser R, Zilker A, Radermacher M, Gaub H, Hartmann T, Baumeister W . Interfacial energies and surface-tension forces involved in the preparation of thin, flat crystals of biological macromolecules for high-resolution electron microscopy. J Microsc. 1991; 161(Pt 1):21-45. DOI: 10.1111/j.1365-2818.1991.tb03071.x. View

10.

Varga S, Taylor K, Martonosi A . Effects of solutes on the formation of crystalline sheets of the Ca(2+)-ATPase in detergent-solubilized sarcoplasmic reticulum. Biochim Biophys Acta. 1991; 1070(2):374-86. DOI: 10.1016/0005-2736(91)90078-m. View

11.

Lacapere J, Stokes D, Chatenay D . Atomic force microscopy of three-dimensional membrane protein crystals. Ca-ATPase of sarcoplasmic reticulum. Biophys J. 1992; 63(2):303-8. PMC: 1262152. DOI: 10.1016/S0006-3495(92)81600-4. View

12.

Bigelow D, Inesi G . Contributions of chemical derivatization and spectroscopic studies to the characterization of the Ca2+ transport ATPase of sarcoplasmic reticulum. Biochim Biophys Acta. 1992; 1113(3-4):323-38. DOI: 10.1016/0304-4157(92)90005-u. View

13.

Glaeser R . Specimen flatness of thin crystalline arrays: influence of the substrate. Ultramicroscopy. 1992; 46(1-4):33-43. DOI: 10.1016/0304-3991(92)90006-6. View

14.

Green N . Evolutionary relationships within the family of P-type cation pumps. Ann N Y Acad Sci. 1992; 671:104-12. DOI: 10.1111/j.1749-6632.1992.tb43788.x. View

15.

Toyoshima C, Sasabe H, Stokes D . Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplasmic reticulum membrane. Nature. 1993; 362(6419):467-71. DOI: 10.1038/362469a0. View

16.

Leapman R, Brink J, Chiu W . Low-dose thickness measurement of glucose-embedded protein crystals by electron energy loss spectroscopy and STEM dark-field imaging. Ultramicroscopy. 1993; 52(2):157-66. DOI: 10.1016/0304-3991(93)90186-2. View

17.

Kuhlbrandt W, Wang D, Fujiyoshi Y . Atomic model of plant light-harvesting complex by electron crystallography. Nature. 1994; 367(6464):614-21. DOI: 10.1038/367614a0. View

18.

Taylor K, Varga S . Similarity of three-dimensional microcrystals of detergent-solubilized (Na+,K+)-ATPase from pig kidney and Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum. J Biol Chem. 1994; 269(13):10107-11. View

19.

Stokes D, Taylor W, Green N . Structure, transmembrane topology and helix packing of P-type ion pumps. FEBS Lett. 1994; 346(1):32-8. DOI: 10.1016/0014-5793(94)00297-5. View

20.

Inesi G . Teaching active transport at the turn of the twenty-first century: recent discoveries and conceptual changes. Biophys J. 1994; 66(3 Pt 1):554-60. PMC: 1275754. DOI: 10.1016/s0006-3495(94)80872-0. View