Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM

Area of Science:

• Structural Biology
• Biochemistry
• Microscopy

Background:

• Cryo-electron microscopy (cryoEM) is vital for high-resolution biomolecular structure determination.
• Sample preparation, especially for low-abundance or difficult proteins, remains a significant challenge in cryoEM.
• Preferential orientation and air-water interface exposure can hinder data quality.

Purpose of the Study:

• To develop an improved cryoEM sample preparation method for challenging biomolecules.
• To enhance particle absorption and minimize air-water interface effects.
• To determine the structure of the Thermococcus kodakarensis mini-chromosome maintenance complex 3 (MCM3) and its complexes.

Main Methods:

• Developed an affinity-grid method using streptavidin on a lipid monolayer.
• Employed biotinylation to enrich MCM3 and MCM3-ATP-dsDNA complexes.
• Utilized cryo-electron microscopy for structure determination.

Main Results:

• Successfully enriched and determined the structure of MCM3 using the affinity-grid method.
• Resolved the structure of a stable MCM3-ATP-dsDNA complex.
• Observed novel left-handed open spiral conformations for both MCM3 apo and dsDNA-bound states.
• Demonstrated the method's utility with HIV-1 capsid protein and RNA polymerase II elongation complex, mitigating preferential orientation issues.

Conclusions:

• The developed affinity-grid method significantly enhances cryoEM sample preparation for challenging targets.
• The novel MCM3 structures provide new insights into its conformational states and DNA binding/melting potential.
• This method is broadly applicable for improving cryoEM studies of various protein complexes.