Electron microscope observations on Ca2+-ATPase microcrystals in detergent-solubilized sarcoplasmic reticulum

Area of Science:

• Biochemistry
• Structural Biology
• Membrane Proteins

Background:

• Sarcoplasmic reticulum Ca2+-ATPase is crucial for muscle contraction.
• Understanding its structure is key to elucidating its function.
• Previous studies faced challenges in crystallizing membrane proteins.

Purpose of the Study:

• To develop a method for crystallizing Ca2+-ATPase molecules.
• To characterize the structure of these Ca2+-ATPase crystals.
• To investigate the stability and protective mechanisms of the crystals.

Main Methods:

• Incubation of detergent-solubilized sarcoplasmic reticulum under controlled conditions.
• Electron microscopy techniques including negative staining, freeze-fracture, and cryo-hydration.
• Crystallographic analysis of lattice dimensions.
• Cross-linking with glutaraldehyde for stability assessment.

Main Results:

• Formation of crystalline arrays of Ca2+-ATPase molecules in stacked lamellar structures.
• Identification of a centered rectangular lattice with dimensions 164 x 55.5 Å.
• Demonstration that glycerol and high Ca2+ concentrations promote crystal formation.
• Glutaraldehyde cross-linking significantly enhances crystal stability against various disruptive agents.

Conclusions:

• A reproducible method for Ca2+-ATPase crystallization was established.
• The study reveals the detailed structural organization of Ca2+-ATPase in crystalline arrays.
• Glutaraldehyde fixation provides a robust method for preserving Ca2+-ATPase crystal structures.