The graphene-based affinity cryo-EM grid for the endogenous protein structure determination

Area of Science:

• Structural Biology
• Biophysics
• Cryo-Electron Microscopy

Background:

• Sample preparation remains a key challenge for high-resolution cryo-electron microscopy (cryo-EM).
• Targeting low-abundance endogenous protein complexes requires advanced techniques for efficient capture and structural analysis.

Purpose of the Study:

• To develop and validate a novel graphene-based affinity cryo-EM grid, the Graffendor (GFD) grid, for improved structural determination of challenging protein targets.
• To demonstrate the GFD grid's capability in capturing both tagged and endogenous proteins for high-resolution cryo-EM analysis.

Main Methods:

• Development of a one-step crosslinking batch-production method for GFD grids using ALFA nanobodies (GFD-A grid).
• Application of GFD-A grids for capturing tagged β-galactosidase and endogenous yeast proteins (Pop6, RNase MRP, RNase P).
• Cryo-EM data collection and structure determination of target proteins at high resolution.

Main Results:

• The GFD-A grid successfully captured tagged proteins, enabling cryo-EM structure determination at 2.71 Å.
• High-resolution cryo-EM structures of endogenous RNase MRP (3.3 Å from lysate, 3.6 Å from eluate) and RNase P (3.0 Å from lysate, 3.9 Å from eluate) were obtained.
• Additional densities observed in lysate-derived structures suggest the capture of transient interactions, absent in eluate-derived structures.

Conclusions:

• The Graffendor (GFD) grid provides a robust platform for investigating endogenous proteins using cryo-EM.
• This method enhances the efficiency and resolution of cryo-EM studies for challenging targets, including those with transient interactions.
• The GFD-A grid facilitates the structural analysis of low-abundance protein complexes, advancing the field of structural biology.