Hydrogen properties in an organic molecule revealed by XFEL and electron crystallography

Abstract

Structure analysis of small crystals is important in synthetic organic chemistry, pharmaceutical and material sciences, and related areas, as the conformation of these molecules may differ in large and small crystals, thus affecting the interpretation of their functional properties and drug efficacy. From small crystals, X-ray and electron beams could furnish electron densities and Coulomb potentials of target molecules, respectively. The two beams provide distinctly different information, and this potential has not been fully explored. Here we present the detailed structure of an organic molecule, rhodamine-6g by X-ray free-electron laser (XFEL) and electron crystallography from the same sample batch of microcrystals. This is the first organic molecular structure determined using XFEL at subatomic resolution. Direct comparison between the electron-density and the Coulomb-potential maps together with theoretical models based on Poisson’s equation shows that the position of hydrogen atoms depends on bond type and charge distribution. The combined approach could lead to better insights into their chemical and/or binding properties for a broad range of organic molecules.

Hydrogen properties and charges in a sub-1.2 Å resolution cryo-EM structure revealed by a cold field emission beam

“…The electron beam makes the hydrogen densities appear even slightly further from the bonded atom than the nucleus of hydrogen (3,27). We have shown that in the Coulomb potential map of an organic molecule the hydrogen density peak position depends on the type of covalent bond (27). There is a larger change in the position of the hydrogen peaks in electron density maps depending on the polarity of bonding due to the mobility of electrons (29,30,31).…”

“…In single particle reconstructions and Coulomb potential maps by 3D ED, hydrogen densities appear further away from their parent atoms than those in X-ray crystal structures (28,3,27). This reflects the fact that incident electrons are affected by both electrons and nuclear charges and the latter is dominant, while X-rays are scattered by electrons around atoms and the electron in a hydrogen atom is attracted towards its parent bonded atom (29,30).…”

“…Difference maps could provide smaller differences between the experimental data and model, but our recent 3D ED study showed that interpretation of residual densities in a difference map is more challenging due to distributions of partial charges and suboptimal assignment of electron scattering factors to the corresponding model (27). Conversion to electron density may provide a more interpretable result (27). Still, further work is needed for a more extensive analysis of charges.…”

“…This reflects the fact that incident electrons are affected by both electrons and nuclear charges and the latter is dominant, while X-rays are scattered by electrons around atoms and the electron in a hydrogen atom is attracted towards its parent bonded atom (29,30). The electron beam makes the hydrogen densities appear even slightly further from the bonded atom than the nucleus of hydrogen (3,27). We have shown that in the Coulomb potential map of an organic molecule the hydrogen density peak position depends on the type of covalent bond (27).…”

“…There is a larger change in the position of the hydrogen peaks in electron density maps depending on the polarity of bonding due to the mobility of electrons (29,30,31). In general, the higher the attractive force shortens the bond length (29,27), while conversely higher B-factors and lower resolution lengthen the apparent distance (3).…”