Two-dimensional crystallization of integral membrane proteins for electron crystallography

Area of Science:

• Structural biology
• Biochemistry
• Electron microscopy

Background:

• Membrane proteins constitute 30% of the proteome and are crucial therapeutic targets.
• Determining the atomic structure of membrane proteins is challenging using conventional methods like X-ray diffraction and NMR.
• Electron crystallography provides an alternative approach for membrane protein structure determination.

Purpose of the Study:

• To review methods for producing two-dimensional crystals of membrane proteins.
• To describe screening techniques for these crystals using negative stain electron microscopy.
• To present image analysis procedures for evaluating crystal quality for cryoelectron microscopy.

Main Methods:

• Reconstitution of purified, detergent-solubilized membrane proteins into lipid bilayers.
• Formation of two-dimensional crystals within these reconstituted systems.
• Screening of crystal quality and morphology using negative stain electron microscopy.

Main Results:

• Examples of various common two-dimensional crystal morphologies are presented.
• Basic image analysis procedures for assessing crystal suitability are described.
• The review highlights the potential of electron crystallography for membrane protein structure determination.

Conclusions:

• Electron crystallography is a viable alternative for determining membrane protein structures.
• The methods discussed facilitate the production and evaluation of two-dimensional crystals.
• This approach aids in advancing the structural analysis of these important biomolecules.