Electron 3D Crystallography of Thin Protein Crystals: Atomic Model with Charges

Koji Yonekura, Kazuyuki Kato, Mitsuo Ogasawara, Masahiro Tomita and Chikashi Toyoshima

Abstract: Membrane proteins and biological macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam and X-ray free electron laser. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons 4 - 5 orders of magnitude more strongly than they do X-rays. Another important feature of electron scattering is that the diffraction pattern formed by elastically scattered electrons is directly related to the distribution of Coulomb potential. Thus, electron crystallography could provide a unique method to visualize the charged states of amino acid residues and metals. We have developed a methodology for electron crystallography of ultra-thin (only a few layers thick) 3D protein crystals and presented the Coulomb potential maps at 3.4 Å and 3.2 Å resolution, respectively, obtained from Ca²⁺-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca²⁺-binding sites of Ca²⁺-ATPase and that of the iron atom in the heme in catalase. Here we report the development and first results, and briefly discuss future applications toward structure determination with charges.

Key words: Electron crystallography, 3D crystals, Charge information, Cryo-EM, Protein structure analysis