A history of protein crystallography is looked back over for the past forty years where out-standing methodological development has been made. Protein crystallography has become one of well matured experimental techniques and made a great contribution to remarkable progress in the field of structural biology. Particularly in advance of the last decade, ultrahigh resolution structure analyses of protein crystals enabled us to visualize distribution of outer-electron density which implies that new era to understand chemistry of protein reaction from crystal structures will come soon.

Labile chemical bonds generate an intermediate state of chemical reaction and exhibit an ultimate of orbital interaction forming a chemical bond. Detailed investigation of such bonds construct a solid basis for understanding the origin of chemical bond and bond formation. Herein the author describes electronic structures of labile chemical bonds such as hypervalent bonds, small-cyclic alkyne, and intermediate states of C-C bond cleavage, derived by experimental X-ray electron density distribution（EDD）analysis. A novel approach is also presented for analyzing the mechanism of catalytic reaction based on EDD analysis closely related to spectroscopy and theoretical calculations.

Molecular orbitals were analyzed successfully by the X-ray molecular orbital analysis（XMO）. Historical background and the bright future of X-ray electron density analysis based on wavefunctions are described including the basic framework of the method.

X-ray charge density is one of the most information rich observables in natural science. We have developed the diffractometers and measurement techniques for X-ray charge density study at one of the third generation synchrotron radiation source SPring-8. Very small amounts of aspherical electron distribution of a pure metal aluminum were observed in the static deformation density. A signal to noise ratio of high temperature powder diffraction data was improved by the data correction based on Fourier analysis.

Hirshfeld atom refinement （HAR） is a method which determines structural parameters from single-crystal X-ray diffraction data by using an aspherical atom partitioning of tailor-made ab initio quantum mechanical molecular electron densities. In this article, a short introduction of the method and procedures are presented along with an actual example of the HAR refinement and evaluations of the result.