Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular...

Area of Science:

• Structural Biology
• Biophysics
• Microscopy

Background:

• Single particle cryo-electron microscopy (cryo-EM) is vital for macromolecular assembly structural studies.
• Studying low-abundance, low-yield, or short-lived samples remains a challenge in cryo-EM.
• Affinity grid techniques offer potential solutions by integrating sample purification and grid preparation.

Purpose of the Study:

• To introduce and validate cryo-SPIEM, a novel affinity cryo-EM approach.
• To demonstrate cryo-SPIEM's utility for studying challenging biological samples.
• To provide a simplified and user-friendly method for cryo-EM grid preparation.

Main Methods:

• Development of cryo-SPIEM, adapting Solid Phase Immune Electron Microscopy (SPIEM) for cryo-EM.
• Application of cryo-SPIEM to various native macromolecular complexes using established antibodies.
• Testing cryo-SPIEM with diverse samples: His-tagged bacteriophage T7, E. coli ribosomes, Sindbis virus, and Tulane virus.

Main Results:

• Cryo-SPIEM successfully trapped and concentrated target particles on TEM grids.
• Minimal view constraints were observed, facilitating cryo-EM imaging.
• The method proved effective for high/low molecular weight, high/low symmetry, His-tagged, and native particles.

Conclusions:

• Cryo-SPIEM is a versatile and effective affinity grid technique for cryo-EM.
• This approach enables structural studies of native samples directly from cell cultures.
• Cryo-SPIEM expands the applicability of cryo-EM to challenging and low-abundance biological targets.