Chemical bonding effects in the determination of protein structures by electron crystallography

Scattering of electrons is affected by the distribution of valence electron s that participate in chemical bonding and thus change the electrostatic sh ielding of the nucleus. This effect is particularly significant for low-ang le scattering. Thus, while chemical bonding effects are difficult to measur e with small-unit cell materials, they can be substantial in the study of p roteins by electron crystallography. This work investigates the magnitude o f chemical bonding effects for a representative collection of protein fragm ents and a model ligand for nucleotide-binding proteins within the resoluti on range generally used in determining protein structures by electron cryst allography. Electrostatic potentials were calculated by ab initio methods f or both the test molecules and for superpositions of their free atoms. Diff erences in scattering amplitudes can be well over 10% in the resolution ran ge below 5 Angstrom and are especially large in the case of ionized side ch ains and ligands. We conclude that the use of molecule-based scattering fac tors can provide a much more accurate representation of the low-resolution data obtained in electron crystallographic studies. The comparison of neutr al and ionic structure factors at resolutions below 5 Angstrom, can also pr ovide a sensitive determination of charge states, important for biological function, that is not accessible from X-ray crystallographic measurements.