Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins

Area of Science:

• Structural Biology
• Biochemistry
• Microscopy

Background:

• Single-particle cryo-electron microscopy (cryoEM) is crucial for atomic resolution studies.
• Current cryoEM methods primarily focus on aerobic samples, limiting the study of air-sensitive biological systems.
• Air-sensitive enzymes and microorganisms are vital yet understudied in structural biology.

Purpose of the Study:

• To develop and detail an anaerobic workflow for single-particle cryoEM.
• To enable high-resolution structural determination of oxygen-sensitive proteins, specifically nitrogenase enzymes.
• To provide a protocol adaptable for other air-sensitive biological samples.

Main Methods:

• Utilized Schlenk lines and anaerobic chambers for sample preparation under an inert atmosphere.
• Incorporated a protein tag to monitor oxygen exposure.
• Adapted a plunge freezing apparatus within a specialized anaerobic chamber for cryoEM grid preparation.
• Implemented a custom vacuum port for safe evacuation of flammable vapors.

Main Results:

• Successfully obtained high-resolution structures of nitrogenase proteins using the developed anaerobic cryoEM workflow.
• Demonstrated the efficacy of the protocol for characterizing air-sensitive enzymes.
• Established a comprehensive protocol involving anaerobic sample generation and cryoEM preparation.

Conclusions:

• The developed anaerobic cryoEM workflow successfully overcomes limitations in studying air-sensitive biological systems.
• This methodology significantly advances the structural biology of enzymes like nitrogenase.
• The protocol is adaptable for various controlled gas environments and oxygen-sensitive proteins.