Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins

Area of Science:

• Biophysics
• Structural Biology
• Biochemistry

Background:

• Preparing cryoelectron microscopy (cryo-EM) grids for amyloid fibril imaging, such as human islet amyloid polypeptide (hIAPP), is challenging.
• Existing methods often require nonphysiological conditions, tags, or seeding, limiting structural studies of fibrils formed under diverse conditions.
• Efficient and reproducible grid preparation is crucial for elucidating amyloid fibril structures.

Purpose of the Study:

• To develop improved cryoelectron microscopy (cryo-EM) grid preparation techniques for amyloid fibrils.
• To identify solution compositions that enhance fibril distribution and ice stability for imaging.
• To determine the structure of the dominant hIAPP polymorph formed in vitro at physiological pH.

Main Methods:

• Systematic evaluation of buffer compositions, focusing on physiological pH, for cryo-EM grid preparation of hIAPP.
• Investigation of the role of solution composition in stabilizing thin vitreous ice films.
• Utilizing a nonamyloidogenic rat IAPP (rIAPP) as a 'spectator protein' to improve particle coverage and ice quality.

Main Results:

• HEPES buffer was identified as uniquely enhancing hIAPP fibril distribution and ice layer stability at physiological pH.
• HEPES directly interacts with hIAPP, reducing aggregation and preventing ice nucleation.
• Addition of rIAPP improved grid coverage and ice quality, acting as a surfactant and disentangling hIAPP clusters.
• The structure of the dominant hIAPP polymorph at pH 7.4 was resolved to 4 Å resolution.

Conclusions:

• Solution composition critically influences fibril distribution and ice stability in cryo-EM grid preparation.
• HEPES buffer and the addition of rIAPP represent significant advances in preparing amyloid fibril grids.
• These strategies enable structural determination of amyloid proteins previously resistant to conventional methods.