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. 2019 Apr 23;10(1):1864.
doi: 10.1038/s41467-019-09836-0.

A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold

Yuxi Liu  1   2   3 , Duc T Huynh  1 , Todd O Yeates  4   5   6   7
Affiliations

A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold

Yuxi Liu et al. Nat Commun. .

Abstract

Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this powerful structural technique. Here we use a designed protein scaffold to bind and symmetrically display 12 copies of a small 26 kDa protein, green fluorescent protein (GFP). We show that the bound cargo protein is held rigidly enough to visualize it at a resolution of 3.8 Å by cryo-EM, where specific structural features of the protein are visible. The designed scaffold is modular and can be modified through modest changes in its amino acid sequence to bind and display diverse proteins for imaging, thus providing a general method to break through the lower size limitation in cryo-EM.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The design of a modular protein scaffold for cryo-EM imaging. The designed core assembly is composed of 12 copies of two protein subunits, A (yellow) and B (orange), in a tetrahedrally symmetric arrangement. Subunit A is genetically fused by a continuous alpha helical linker to a DARPin (green). Amino acid mutations (based on random library selection experiments) are inserted into the DARPin binding surface (pink) to confer tight binding of a cargo protein (blue) for cryo-EM imaging
Fig. 2
Fig. 2
Cryo-EM data on GFP bound scaffolds. a 2D class images of the particles show strong features for the tetrahedral core along with clear but more diffuse density for the DARPin and the bound GFP cargo. The image box size in a corresponds to 271 Å. The 3-D density reconstructed only around the core structure (b) and the corresponding gold-standard Fourier shell correlation (FSC) curves (c) are shown for masked (blue) and unmasked (black) maps
Fig. 3
Fig. 3
Near-atomic resolution map of the DARPin adaptor and its bound cargo protein GFP. a A density map obtained following multi-body refinement in RELION is shown colored by local resolution in a side view (left) and a sliced view (right), showing the central helix in the center of the GFP beta barrel. b Gold-standard FSC curve for the reconstruction. c Two wall-eyed stereo views of the sharpened GFP density map. The full GFP density is shown at the top; a sliced view with a fitted GFP atomic model is shown at the bottom. The fluorophore and Leu 64 (shown in sticks) in the central helical region have clear surrounding density. d Comparison between the DARPin plus GFP model in this study (GFP in green, DARPin in magenta) and the previously reported 3G124 eGFP crystal structure (grey, PDB 5MA8). The two structures are aligned on the DARPin portion. e Wall-eyed stereo views of the extensive interface between the DARPin (magenta) and GFP (green). The side chains of the bulky residues forming the interface are shown in sticks
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