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Structural resolution of a small organic molecule by serial X-ray free-electron laser and electron crystallography

Nature Chemistry volume 15, pages 491–497 (2023)Cite this article

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Abstract

Structure analysis of small crystals is important in areas ranging from synthetic organic chemistry to pharmaceutical and material sciences, as many compounds do not yield large crystals. Here we present the detailed characterization of the structure of an organic molecule, rhodamine-6G, determined at a resolution of 0.82 Å by an X-ray free-electron laser (XFEL). Direct comparison of this structure with that obtained by electron crystallography from the same sample batch of microcrystals shows that both methods can accurately distinguish the position of some of the hydrogen atoms, depending on the type of chemical bond in which they are involved. Variations in the distances measured by XFEL and electron diffraction reflect the expected differences in X-ray and electron scatterings. The reliability for atomic coordinates was found to be better with XFEL, but the electron beam showed a higher sensitivity to charges.

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Fig. 1: SX of rhodamine-6G crystals.
Fig. 2: Structures of rhodamine-6G determined by SX and ED.
Fig. 3: 2D slices of electron density and Coulomb potential maps at the plane of the xanthene ring.
Fig. 4: Peak positions of hydrogen densities.

Data availability

Crystallographic data have been deposited at the Cambridge Crystallographic Data Centre, under deposition nos. CCDC 2119567 (SX), 2180418 (rt-ED), 2180417 (cryo-ED) and 2180416 (triclinic-cryo-ED). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/. SX image data have been deposited at the Coherent X-ray Imaging Database (CXIDB), under deposition no. 206 (https://www.cxidb.org/id-206.html). ED image data have been deposited at Zenodo (https://doi.org/10.5281/zenodo.6684913)51. Please also refer to the supporting README document for using the raw image data. Source data are provided with this paper.

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Acknowledgements

We thank K. Hata for designing a special sample–pin mounter, Y. Kageyama for support with sample preparation, K. Hirata for advice on SX data analysis at the beginning of this study, and D. B. McIntosh for help in improving the manuscript. This work was partly supported by JSPS KAKENHI (grant no. 20K15764 to K. Takaba), the JST-Mirai Program (grant no. JPMJMI20G5 to K.Y.), JST CREST (grant no. JPMJCR18J2 to K.Y., S.M.-Y., K. Takaba) and the Cyclic Innovation for Clinical Empowerment (CiCLE) from the Japan Agency for Medical Research and Development, AMED (K.Y.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. Tasuku Hamaguchi

    Present address: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Aoba-ku, Japan

  2. These authors contributed equally: Kiyofumi Takaba, Saori Maki-Yonekura.

Authors and Affiliations

  1. RIKEN SPring-8 Center, Sayo, Hyogo, Japan

    Kiyofumi Takaba, Saori Maki-Yonekura, Ichiro Inoue, Kensuke Tono, Tasuku Hamaguchi, Keisuke Kawakami, Hisashi Naitow, Tetsuya Ishikawa, Makina Yabashi & Koji Yonekura

  2. Japan Synchrotron Radiation Research Institute, Sayo, Hyogo, Japan

    Kensuke Tono & Makina Yabashi

  3. Advanced Electron Microscope Development Unit, RIKEN-JEOL Collaboration Center, RIKEN Baton Zone Program, Sayo, Hyogo, Japan

    Koji Yonekura

  4. Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Aoba-ku, Japan

    Koji Yonekura

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Contributions

K. Takaba, S.M.-Y., I.I., K. Tono, T.I., M.Y. and K.Y. conceived the project. K. Takaba and S.M.-Y. prepared target specimens for SX and ED experiments. I.I., K. Tono and M.Y. set up the XFEL beamline for measurements. K.Y. set up the cryo-electron microscope for measurements. K. Takaba, S.M.-Y. and K.Y. collected SX data, and K. Takaba and S.M.-Y. collected ED data. T.H., K.K. and H.N. supported SX data collection. K. Takaba processed the raw data, solved structures and analysed them. K. Takaba, S.M.-Y., I.I., K. Tono and K.Y. discussed the results. K. Takaba and K.Y. wrote the manuscript. All authors joined in discussions of the manuscript.

Corresponding author

Correspondence to Koji Yonekura.

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The authors declare no competing interests.

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Peer review information

Nature Chemistry thanks Tim Gruene, Daniel Paley and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Atomic models of rhodamine-6g obtained in this study.

(a–c) The SX model determined from the orthorhombic crystal placed in the unit cell (a), the rt-ED (b) and the cryo-ED (c) models from the orthorhombic crystal. (d) The atomic model in (a) with labels of non-hydrogen atoms. (e) Overlay of the models in (a) and (b) along with the unit cells. (f) The triclinic cryo-ED model from the recrystallized crystal. (g) The model in (f) with labels of non-hydrogen atoms. (h) Rhodamine-6g dimers in the orthorhombic (orange) and triclinic crystals (cyan). The rt-ED (b) and triclinic-cryo-ED (f) models are overlaid by adjusting the monomers at the top onto each other.

Extended Data Fig. 2 Similarities among the atomic models of the SX and ED structures.

(a–c) Relation plots between the SX and ED structures in 1,2- (a) and 1,3- (b) bond distances for the same pair of non-hydrogen atoms and in B-factors (c) for the same non-hydrogen atoms. A linear regression is drawn for the rt-ED in (c). See also Supplementary Table 1 for bond lengths between non-hydrogen atoms. Correlation coefficients between the SX and ED data are also shown in the graphs.

Source data

Extended Data Fig. 3 Bond deviations in the atomic models of the SX, ED structures from the reported SCXRD structure.

A histogram of deviations in bond distances between the SCXRD and SX or ED structures, |dSX or ED - dSCXRD|. Bond distances, d, are measured for 1,3- pairs. The root mean square (rms) values of the deviation are represented in the inset of the graphs.

Source data

Extended Data Fig. 4 Peak positions of hydrogen densities in methyl and methylene groups.

(a) The same density plots as in Fig. 4b, c but in C-H3 (methyl) bonds from the SX and rt-ED structures in green and yellow, respectively. (b) The same plots as in (a) but in C-H2 (methylene) bonds. Gray horizontal lines refer to a density level of 1.5σ, and vertical lines represent the positions of the hydrogen nuclei, obtained by ND studies25.

Source data

Extended Data Fig. 5 Diagrams of R value variations for refinement of the rt-ED structure with the charged-atom model.

(a–i) Charge values were varied for specific atoms shown in the horizontal and vertical axes, and R values are represented in gradient colors according to the gradient bar on the right in each diagram. R values for the data with Fo > 4σ in the whole resolution shells (a, d, g), in the lowest resolution shell of s < 0.2 Å−1 (b, e, h) and in the other remaining shells (s ≥ 0.2 Å−1) (c, f, i). Amide-hydrogen atoms (H15 and H16) were exclusively charged with given values along the axes in (a – c). Given a charge of -0.9 to a chloride atom (CL1), H15 and H16 were positively charged in (d – f). Given +0.2 to H16, H15 was positively and CL1 was negatively charged in (g – i). The lowest R value is shown with yellow markers in a, b, d, e, g and h. All possible combinations of charges for H15, H16 and CL1 were examined as in Methods.

Extended Data Fig. 6 Diagrams of R value variations for refinement of the SX structure with the charged-atom model.

(a–i) Done in the same way as in Extended Data Fig. 5, but for the SX data and model. No grid points giving R value minima are depicted and variations in R values are smaller than those for the ED data (c.f. Extended Data Fig. 5).

Extended Data Fig. 7 Configuration of the xanthene ring and the ethoxycabonyl tail in the SX structure of rhodamine-6g.

(a) The plane including the xanthene ring shown in pale pink is labeled ‘α’. The plane ‘β’ in magenta is defined as a cross-section cutting through the center of the xanthene ring along O13 – C6 and perpendicular to the plane α. (b) A side view of (a). The ether oxygen (O31) in the ethoxycarbonyl tail faces to the xanthene-ring side and is close to the central plane β: only 0.163 Å and 0.038 Å apart from the plane in the SX- and cryo-ED structures, respectively. The distances of which are also listed in Supplementary Table 1.

Extended Data Fig. 8 Relation plots between the neutral and charged models for the rt-ED structure.

(a, b) Relation plots between the neutral and charged rt-ED structures in 1,2- bond distances (a) and in B-factors (b) for the same non-hydrogen atoms. Correlation coefficients are shown in the legend.

Source data

Supplementary information

Supplementary Information

Supplementary Discussion, Tables 1–6, Figs. 1 and 2, References and ORTEP drawing of crystal structures.

Supplementary Data 1

Crystallographic data of SX.

Supplementary Data 2

Crystallographic data of rtED.

Supplementary Data 3

Structure factors for rtED.

Supplementary Data 4

Crystallographic data of cryoED.

Supplementary Data 5

Structure factors for cryoED.

Supplementary Data 6

Crystallographic data of triclinic-cryoED.

Supplementary Data 7

Structure factors for triclinic-cryoED.

Supplementary Data 8

README document source for data processing.

Supplementary Data 9

Source Data for Supplementary Fig. 1c.

Supplementary Table 1

A workbook of Supplementary Tables 1–6.

Source data

Source Data Fig. 1

Geometrical statistics of measured diffraction data.

Source Data Fig. 2

Geometrical statistics of molecular structure data.

Source Data Fig. 4

Geometrical features of molecular density data.

Source Data Extended Data Fig./Table 2

Geometrical statistics of molecular structure data.

Source Data Extended Data Fig./Table 3

Geometrical statistics of molecular structure data.

Source Data Extended Data Fig./Table 4

Geometrical features of molecular density data.

Source Data Extended Data Fig./Table 8

Geometrical statistics of molecular structure data.

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Takaba, K., Maki-Yonekura, S., Inoue, I. et al. Structural resolution of a small organic molecule by serial X-ray free-electron laser and electron crystallography. Nat. Chem. 15, 491–497 (2023). https://doi.org/10.1038/s41557-023-01162-9

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