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

Abstract

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 signi®cant for lowangle scattering. Thus, while chemical bonding effects are dif®cult 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 Ê 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 Ê can also provide a sensitive determination of charge states, important for biological function, that is not accessible from X-ray crystallographic measurements.

On the interpretation of electron microscopic maps of biological macromolecules

“…For that reason, the redistributions of valence electrons associated with bond formation, which are likely to alter the spherically averaged X‐ray structure factor of an atom somewhat, but more important, make the non‐spherically averaged X‐ray structure factor of that atom far more anisotropic than it otherwise would be, have a comparatively large impact on electron structure factors at low‐resolution. Thus the effects of bonding will be larger in EP maps that include low resolution data than they are in EP maps computed using only data collected at high‐resolution, where nuclear charges dominate scattering factors …”

“…Atomic charges must be taken into account if a reasonable level of agreement between experimental EP maps and model maps is to be achieved. None of this should come as a surprise . The reason for presenting these observations here is to stimulate the biological EM community to pay more attention to these issues going forward.…”

“…Thus the effects of bonding will be larger in EP maps that include low resolution data than they are in EP maps computed using only data collected at high-resolution, where nuclear charges dominate scattering factors. 12,13 Effects of charge on base pairing geometry and atomic positions…”

“…None of this should come as a surprise. 12,13 The reason for presenting these observations here is to stimulate the biological EM community to pay more attention to these issues going forward.…”

Water Structure from Scattering Experiments and Simulation

“…Deviations from the independent atom model due to chemical bonding effects have been known and studied for many years in the small molecule x-ray diffraction literature 78,97,98 , and more recently in solution scattering 41,57 , and in the context of appropriate form factors for electron crystallography 99,100 . We have recently introduced a modification of the atomic scattering factors for liquid water which rescales them properly at low Q where chemical bonding effects are known to be significant, while retaining their values at large Q where chemical bonding effects on the core electron distribution should be negligible 41 .…”