Has negative staining still a place in biomacromolecular electron microscopy?

Area of Science:

• Structural biology
• Biophysics
• Biochemistry

Background:

• Transmission electron microscopy (TEM) has been instrumental in determining protein structures at molecular resolution.
• Near-atomic resolution data has been achieved for some protein samples using TEM.
• Understanding the capabilities and limitations of TEM is crucial for structural studies.

Purpose of the Study:

• To critically evaluate the potential and limitations of TEM for obtaining molecular resolution structures of proteins.
• To compare the resolution achievable with negatively stained TEM specimens versus cryomicroscopy techniques.
• To provide perspective on the current state of high-resolution protein structure determination using electron microscopy.

Main Methods:

• Critical review of existing literature on transmission electron microscopy of proteins.
• Analysis of data obtained from negatively stained protein specimens.
• Comparison with data from cryomicroscopy of unstained frozen-hydrated and sugar-embedded protein preparations.

Main Results:

• Negatively stained TEM can provide molecular-resolution structural information for proteins.
• Achieving near-atomic resolution with negatively stained specimens has limitations.
• Cryomicroscopy of unstained frozen-hydrated and sugar-embedded samples offers alternative routes to high-resolution structures.

Conclusions:

• TEM, particularly with negative staining, offers valuable molecular insights into protein structures.
• Limitations exist in achieving the highest resolutions with negative staining compared to cryo-EM techniques.
• Cryomicroscopy provides complementary and often superior methods for high-resolution protein structure determination.