GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM.

Area of Science:

• Biochemistry
• Structural Biology
• Microscopy

Background:

• Membrane protein complex preparation for cryo-electron microscopy (cryo-EM) is challenging due to detergent stabilization.
• Achieving monodisperse and stable complexes is crucial for high-resolution structure determination.

Purpose of the Study:

• To develop a novel method, GraDeR, for improved preparation of membrane protein complexes for single-particle cryo-EM.
• To demonstrate the efficacy of GraDeR on various membrane complexes, including mammalian FoF1 ATP synthase.

Main Methods:

• GraDeR utilizes glycerol gradient centrifugation for mild removal of detergent monomers and micelles.
• This process stabilizes lauryl maltose-neopentyl glycol detergent-solubilized membrane complexes.
• Standard cryo-EM protocols for water-soluble proteins can then be applied.

Main Results:

• GraDeR successfully produced monodisperse and stable membrane complexes from three different examples.
• The method allowed visualization of the F1 domain asymmetry in the mammalian FoF1 ATP synthase.
• The observed asymmetry aligns with the ground state structure of the isolated F1 domain.

Conclusions:

• GraDeR significantly improves membrane protein complex preparation for cryo-EM.
• The cryo-EM structure of FoF1 ATP synthase provides direct evidence for Boyer's binding change mechanism in the intact enzyme.
• This work facilitates structural studies of dynamic membrane protein machines.