An efficient clear-native PAGE-based workflow for cryo-electron microscopy sample preparation of large protein...

Area of Science:

• Biochemistry
• Structural Biology
• Microscopy

Background:

• Cryo-electron microscopy (cryo-EM) enables high-resolution protein structure determination.
• Preparing ultra-large protein complexes (>700 kDa) for cryo-EM is challenging, requiring preservation of native structure and integrity.
• Existing methods like sucrose density gradient centrifugation are inefficient, while native PAGE lacks established extraction protocols for cryo-EM.

Purpose of the Study:

• To develop a rapid, efficient cryo-EM sample preparation method for ultra-large protein complexes.
• To optimize electroelution from native polyacrylamide gel electrophoresis (native PAGE) using a specialized buffer system.
• To overcome purification challenges for previously inaccessible protein complexes in cryo-EM.

Main Methods:

• Developed an agarose-acrylamide composite gel for clear-native PAGE (CN-PAGE), offering improved mechanical robustness and ease of band excision.
• Utilized electroelution with an optimized buffer system to recover protein complexes from the gel.
• Employed ultrafiltration for buffer exchange to remove sodium deoxycholate (DOC) prior to grid preparation.

Main Results:

• Achieved high-resolution (2.18 Å) cryo-EM structure of the photosystem I-light-harvesting complex I (PSI-LHCI) supercomplex.
• The developed method requires minimal purification steps, primarily buffer exchange to remove DOC.
• Demonstrated that DOC removal is critical for successful cryo-EM grid preparation.

Conclusions:

• The novel method efficiently isolates large protein complexes from small sample volumes for cryo-EM.
• This technique expands the range of challenging protein systems amenable to cryo-EM structural analysis.
• The optimized electroelution protocol simplifies sample preparation, overcoming previous purification hurdles.