Antibody-Based Affinity Cryoelectron Microscopy at 2.6-Å Resolution.

Area of Science:

• Structural Biology
• Biophysics
• Microscopy

Background:

• Affinity cryo-electron microscopy (cryo-EM) simplifies sample preparation for cryo-EM.
• This technique aims to analyze challenging specimens, including those of low abundance or unpurified.
• Current limitations include the inability to achieve near-atomic resolution, leading to a perception of it as a niche method.

Purpose of the Study:

• To demonstrate that affinity cryo-EM can achieve near-atomic resolution.
• To solve the structure of a challenging, low-abundance sample using affinity cryo-EM.
• To compare the quality of affinity cryo-EM with conventional methods.

Main Methods:

• Utilized an antibody-based affinity grid approach for sample preparation.
• Applied cryo-EM to analyze low-concentration Tulane virus from a low-yield cell-culture system.
• Performed quantitative analyses to assess data and reconstruction quality.

Main Results:

• Successfully solved a ∼2.6-Å structure of Tulane virus.
• The sample was challenging due to low concentration and low yield, making it difficult for standard cryo-EM.
• Quantitative analyses showed data and reconstruction quality comparable to conventional methods using high-concentration samples.

Conclusions:

• Affinity cryo-EM is capable of achieving near-atomic resolution, even for difficult samples.
• The antibody-based affinity grid approach overcomes limitations of standard cryo-EM for low-abundance specimens.
• This advancement expands the utility of cryo-EM for structural biology research.