High-resolution cryo-EM using a common LaB₆ 120-keV electron microscope equipped with a sub-200-keV direct electron...

Area of Science:

• Structural Biology
• Microscopy Techniques
• Biophysics

Background:

• High-resolution cryo-electron microscopy (cryo-EM) typically necessitates expensive 200-300 keV cryo-transmission electron microscopes (cryo-TEMs).
• Recent advancements in sub-200 keV direct electron detector (DED) technology offer potential for more affordable cryo-TEMs.
• Current 100 keV cryo-EM imaging relies on field emission gun (FEG) sources.

Purpose of the Study:

• To investigate the feasibility of upgrading standard 120 keV TEMs with a thermionic lanthanum hexaboride (LaB₆) source for high-resolution cryo-EM.
• To assess the performance of a sub-200 keV DED in conjunction with a LaB₆ source for cryo-EM structure determination.
• To explore a cost-effective approach for obtaining interpretable cryo-EM structures.

Main Methods:

• Utilized a standard 120 keV TEM equipped with a thermionic LaB₆ source.
• Integrated a sub-200 keV optimized direct electron detector (DED).
• Employed automated data collection using SerialEM with a side-entry cryo-holder.

Main Results:

• Achieved a 2.65 Å resolution reconstruction of apoferritin.
• Obtained a 4.33 Å resolution reconstruction of 64 kDa hemoglobin.
• Resolved an asymmetric 153 kDa membrane protein GPCR at 4.4 Å resolution.

Conclusions:

• Demonstrated that standard 120 keV TEMs with LaB₆ sources can be upgraded for high-resolution cryo-EM.
• Showcased the potential for widely accessible and low-cost cryo-EM structure determination.
• Contributed to the democratization of cryo-EM technology for broader scientific application.