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

Scattering of electrons is affected by the distribution of valence electrons that participate in chemical bonding and thus change the electrostatic shielding of the nucleus. This effect is particularly significant for low-angle scattering. Thus, while chemical bonding effects are difficult to measure with small-unit cell materials, they can be substantial in the study of proteins by electron crystallography. This work investigates the magnitude of chemical bonding effects for a representative collection of protein fragments and a model ligand for nucleotide-binding proteins within the resolution range generally used in determining protein structures by electron crystallography. Electrostatic potentials were calculated by ab initio methods for both the test molecules and for superpositions of their free atoms. Differences in scattering amplitudes can be well over 10% in the resolution range below 5 A and are especially large in the case of ionized side chains and ligands. We conclude that the use of molecule-based scattering factors can provide a much more accurate representation of the low-resolution data obtained in electron crystallographic studies. The comparison of neutral and ionic structure factors at resolutions below 5 A can also provide a sensitive determination of charge states, important for biological function, that is not accessible from X-ray crystallographic measurements.