Structure Determination of Biogenic Crystals Directly from 3D Electron Diffraction Data

Abstract

Highly reflective assemblies of purine, pteridine, and flavin crystals are used in the coloration and visual systems of many different animals. However, structure determination of biogenic crystals by single-crystal XRD is challenging due to the submicrometer size and beam sensitivity of the crystals, and powder XRD is inhibited due to the small volumes of powders, crystalline impurity phases, and significant preferred orientation. Consequently, the crystal structures of many biogenic materials remain unknown. Herein, we demonstrate that the 3D electron diffraction (3D ED) technique provides a powerful alternative approach, reporting the successful structure determination of biogenic guanine crystals (from spider integument, fish scales, and scallop eyes) from 3D ED data confirmed by analysis of powder XRD data. The results show that all biogenic guanine crystals studied are the previously known β-polymorph. This study highlights the considerable potential of 3D ED for elucidating the structures of biogenic molecular crystals in the nanometer-to-micrometer size range. This opens up an important opportunity in the development of organic biomineralization, for which structural knowledge is critical for understanding the optical functions of biogenic materials and their possible applications as sustainable, biocompatible optical materials.

Figure 1

Morphologies of biogenic guanine crystals imaged by TEM. (a) Prismatic guanine crystal doublets extracted from iridophore cells in the integument of the white widow spider (Latrodectus pallidus). Top left: L. pallidus. Top right: higher magnification optical micrograph of the white iridophore cells. (b) Square-plate guanine crystals extracted from the image-forming mirror of scallop eyes. Top left: adult scallop, Pecten maximus. Top right: optical micrograph of part of the mantle tissue with three visible eyes. (c) Elongated hexagonal plate crystals extracted from salmon (Salmo salar) scales. Top left: salmon skin. Top right: optical micrograph of one scale at higher magnification showing the iridescent iridophore cells. In the TEM images in parts a–c, the (100) crystal face was assigned by zone-axis ED carried out during imaging.

Figure 2

Reconstructed precession images from the 3D ED data recorded for a guanine crystal from a spider showing the reciprocal lattice planes reconstructed from the experimental data. In addition, the gray panels correspond to simulated planes (see Section S2) for the crystal structure determined for the spider sample from the 3D ED data in P2₁/c: (a) (0kl), (b) (h0l), and (c) (hk0). The inset in part a shows the crystal from which the data set was collected (scale bar 1 μm).

Figure 3

(a) Electron density map following structure refinement from 3D ED data of guanine crystals from the spider sample showing the successful identification of the hydrogen atom positions, (b) the H-bonded layer parallel to bc-plane of the crystal structure of the β-polymorph, and (c) view along the b-axis showing the H-bonded layers.

Figure 4

Overlay of the relative position of the guanine molecule in the asymmetric unit of the crystal structures determined from 3D ED data for biogenic fish (cyan), spider (yellow), and scallop (magenta) samples and from powder XRD data for the synthetic sample of the β polymorph (green).