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

Abstract

Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this "spectator protein", exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques.

Figure 1

Molecular structures of Tris, MOPS, and HEPES buffers used in the study. To see this figure in color, go online.

Figure 2

Cryo-EM micrographs showing hIAPP fibrils aggregated under different buffer conditions. The top panel presents micrographs at grid holes magnification (8500×) while the bottom panel shows micrographs at acquisition magnification (92,000×). Among all the tested physiological pH buffers, only HEPES buffer yields good ice coverage and a satisfactory distribution of fibrils. While MOPS buffer did not facilitate adequate fibril coverage, lowering the pH to a more acidic level significantly improved both fibril coverage and the ice quality. Scale bars, 1 μm (grid holes) and 100 nm (acquisition scale bar).

Figure 3

Cryo-EM micrographs showing hIAPP aggregated in Tris-HCl followed by buffer exchange to HEPES at final concentrations of 10 and 50 mM. Micrographs were collected at grid square magnification (512×, left) and grid hole magnification (8500×, right). The grid square view reveals a trend shift from predominantly dry holes at low HEPES concentration to a majority of holes with a well-formed ice layer. In both scenarios, clumps of fibrils are still observable. Scale bars, 10 μm (grid squares) and 500 nm (grid holes).

Figure 4

Cryo-EM micrographs of hIAPP aggregated in Tris-HCl with the addition of SDS, n-dodecyl-β-D-maltoside (DDM), cetyltrimethylammonium chloride (CTAC), or rIAPP as additive before grid preparation. Micrographs were collected at grid squares magnification (512×, left), grid holes magnification (8500×, middle), and data acquisition magnification (92,000×, right). The presence of 2 mM SDS led to a thin ice layer in every hole, improving the overall quality of the grid greatly. DDM and CTAC also led to better ice quality but to a lesser extent than SDS, with large fibril clumps still visible. The addition of rIAPP not only improved the ice layer but also showed a better distribution of fibrils and less clumping. Scale bars, 10 μm (grid square), 500 nm (grid holes), and 100 nm (acquisition scale bar).

Figure 5

(A) Density map showing the structure of the predominant polymorph found in hIAPP fibrils aggregated in HEPES (pH 7.4). The N-terminal residues 1–12 are unresolved, reflecting their high flexibility. (B) Illustration of lateral stacking in peptide units with β sheets depicted as ribbons. (C) Hydrophobicity map highlighting the interface between individual protofilaments, with hydrophobic interactions in yellow and hydrophilic in teal. (D) Detailed view of the stabilizing hydrogen bonds between Tyr37 and Ser29. (E) Representation of Asp21 forming ladder-type interactions, stabilizing the stacking of peptide units. (F) Close-up of the density around His18, suggesting the presence of two rotameric conformations at this site. To see this figure in color, go online.