It started with a Cys: Spontaneous cysteine modification during cryo-EM grid preparation

Area of Science:

• Structural Biology
• Biochemistry
• Cryo-Electron Microscopy

Background:

• Cryo-electron microscopy (cryo-EM) is increasingly used for structure determination.
• Protein interactions with the air-water interface (AWI) during grid preparation are poorly understood.
• These AWI interactions can cause protein unfolding and preferred orientation, hindering cryo-EM analysis.

Purpose of the Study:

• To investigate the impact of AWI interactions on protein structure during cryo-EM grid preparation.
• To identify the molecular mechanisms behind protein modification at the AWI.
• To develop strategies to mitigate these detrimental effects.

Main Methods:

• Studied modifications of cysteine residues in HSPD1 and beta-galactosidase.
• Investigated time-dependent cysteine modification.
• Assessed mitigation strategies including rapid grid preparation, DTT addition, and continuous support films.

Main Results:

• Cysteine residues in HSPD1 and beta-galactosidase undergo time-dependent modification, linked to partial unfolding.
• Reactive oxygen species at the AWI are implicated in this oxidation.
• Rapid grid preparation (millisecond timescale) prevents cysteine modification, outrunning the reaction kinetics.

Conclusions:

• Spontaneous cysteine modification and protein unfolding occur at the AWI during cryo-EM grid preparation.
• This modification is likely due to oxidation by reactive oxygen species.
• Accelerated grid preparation is a viable strategy to prevent these artifacts and improve cryo-EM structure determination.