Small LEA proteins mitigate air-water interface damage to fragile cryo-EM samples during plunge freezing

Abstract

Air-water interface (AWI) interactions during cryo-electron microscopy (cryo-EM) sample preparation cause significant sample loss, hindering structural biology research. Organisms like nematodes and tardigrades produce Late Embryogenesis Abundant (LEA) proteins to withstand desiccation stress. Here we show that these LEA proteins, when used as additives during plunge freezing, effectively mitigate AWI damage to fragile multi-subunit molecular samples. The resulting high-resolution cryo-EM maps are comparable to or better than those obtained using existing AWI damage mitigation methods. Cryogenic electron tomography reveals that particles are localized at specific interfaces, suggesting LEA proteins form a barrier at the AWI. This interaction may explain the observed sample-dependent preferred orientation of particles. LEA proteins offer a simple, cost-effective, and adaptable approach for cryo-EM structural biologists to overcome AWI-related sample damage, potentially revitalizing challenging projects and advancing the field of structural biology.

Fig. 1. Cryo-EM sample grid preparation and potential role of LEA proteins on AWI damage mitigation.

Cartoon illustration of the process and outcomes of preparing cryo-EM SPA grids and the hypothesized effects of LEA proteins on sample structural integrity. a Shows a Cryo-EM SPA grid preparation robot alongside a detailed view of a typical holey grid with vitrified ice, which is used for embedding protein samples. b–d Depict different states of protein sample distribution within the vitrified ice. In panel (b), an ideal sample distribution is shown where proteins are evenly dispersed without any structural damage. c Illustrates the typical impact of air–water interface (AWI) damage on protein samples, leading to preferred orientation, complex dissociation, and protein denaturation. d Demonstrates how LEA proteins can mitigate sample damage by forming a barrier at the AWI, which significantly mitigates these damages and preserves protein integrity in vitrified ice.

Fig. 2. High-resolution cryo-EM structure determination of fragile human Polymerase alpha-Primase and Polycomb repressive complex 2 using nematode AavLEA1.

Cryo-EM single-particle analysis of human polymerase α-primase complex (PP), (a–c), and polycomb repressive complex 2 (PRC2), panels (d–f), both with the addition of Nematode AavLEA1. a, d Display representative micrographs and CTFs-cropped up to the ice ring for the complexes both alone and with AavLEA1 added at a 1:40 ratio, highlighting improved sample preservation due to LEA protein addition. Representative micrographs were chosen out of 42 (a) top, 4403 (a) bottom, 5 (d) top, and 2843 (d) bottom. b, e Depict 2D class averages, illustrating defined and consistent particle shapes with visible protein features when AavLEA1 was used. Finally, c, f show reconstructed cryo-EM maps of the complexes from the AavLEA1 datasets, presented in two orientations.

Fig. 3. A truncated form of LEA protein from tardigrade also mitigates AWI sample damage.

Cryo-EM single-particle analysis of protein complexes with RvLEAM_short. a, d Display representative micrographs and CTFs-cropped up to the ice ring-out of the 1308 and 3896 collected respectively of the polymerase-primase complex (PP) and Polycomb repressive complex 2 (PRC2), each treated with RvLEAM_short at a molar ratio of 1:6. b, e Show 2D class averages, which demonstrate the structural homogeneity and quality of the sample with RvLEAM_short added. c, f Present the reconstructed cryo-EM maps of PP and PRC2, depicted in two orientations, achieving resolutions of 4.5 and 3.7 Å, respectively.

Fig. 4. Samples with LEA protein addition distribute at vitrified ice surfaces.

Particle orientation distribution and cryo-electron tomography (cryo-ET) analysis for samples with LEA proteins addition. a Displays Mollweide projections that compare the particle distribution for the polymerase α-primase complex (PP) and Polycomb repressive complex 2 (PRC2) with AavLEA1 (1:40) and RvLEAM_short (1:6) added respectively. Corresponding sphericity values demonstrate the degree of isotropy achieved in the cryo-EM map under each condition. b, c Show cryo-ET cross-sectional analysis of the spatial distribution of PP and PRC2 particles within the grid holes, respectively. These plots highlight the location of particles relative to the edge of the holes and identify regions affected by ice contamination. The axes are expressed in pixels, with a scale of 4.4 Å per pixel.

Fig. 5. Chemical crosslinking enhances the orientation distribution of PRC2 particles in the presence of RvLEAM_short.

a Displays Mollweide projections of the particle orientation distribution for the Polycomb repressive complex 2 (PRC2) treated with chemical crosslinking for 2 and 10 min, demonstrating improved isotropy as evidenced by the increased sphericity values of 0.83 and 0.97, respectively. b Shows the high-resolution cryo-EM maps of PRC2 crosslinked for 10 min, presented in two orientations to highlight the detailed structural features achieved, with a global resolution of 3.1 Å.

Fig. 6. Comparative evaluation of LEA Proteins and CHAPSO as AWI damage mitigation strategies.

a Showcases the reconstructed cryo-EM map of the polymerase α-primase complex, visualized in two orientations, achieving a global resolution of 3.4 Å. b, c Detail the ResLog and per-particle spectra SNR (ppSSNR) analyses respectively, comparing the effects of AavLEA1 and CHAPSO addition on particle image data and map reconstruction quality. The ResLog analysis in panel (b) illustrates the spatial frequency improvements associated with each additive, plotted against batch size on a logarithmic scale, indicating that AavLEA1 outperforms CHAPSO at higher spatial frequencies. c Displays the logarithm of ppSSNR, demonstrating that AavLEA1 maintains higher SNR values across the majority of the spatial frequencies tested. Source data are provided as a Source Data file.