The Affinity Grid: a pre-fabricated EM grid for monolayer purification.

Area of Science:

• Structural Biology
• Biochemistry
• Microscopy

Background:

• Single-particle electron microscopy (EM) requires purified protein specimens.
• Traditional purification methods can be time-consuming and complex.
• His-tagged proteins offer a convenient target for purification.

Purpose of the Study:

• To introduce and characterize the Affinity Grid for simplified specimen preparation.
• To demonstrate the Affinity Grid's utility for purifying various His-tagged complexes.
• To enable rapid structural determination of biological macromolecules.

Main Methods:

• Development of the Affinity Grid: a pre-fabricated EM grid with a Ni-NTA lipid monolayer.
• Application of monolayer purification using the Affinity Grid to cell extracts.
• Isolation of His-tagged ribosomal complexes and membrane proteins.
• 3D reconstruction of ribosomal subunits using vitrified Affinity Grid specimens.

Main Results:

• The Affinity Grid allows rapid isolation of His-tagged ribosomal complexes (e.g., 50S, 70S, 30S subunits) from E. coli extracts.
• Specimen preparation for EM is achieved within minutes.
• His-tagged aquaporin-9 (AQP9) was successfully isolated from membrane fractions.
• High-resolution 3D reconstructions were obtained from the same sample.

Conclusions:

• The Affinity Grid significantly streamlines specimen preparation for single-particle EM.
• This method is versatile, applicable to both soluble complexes and membrane proteins.
• The Affinity Grid facilitates rapid structural analysis of His-tagged biomolecules.