Abstract
Approximately 90% of the structures in the Protein Data Bank (PDB) were obtained by X-ray crystallography or electron microscopy. Whereas the overall quality of structure is considered high, thanks to a wide range of tools for structure validation, uncertainties may arise from density maps of small molecules, such as organic ligands, ions or water, which are non-covalently bound to the biomolecules. Even with some experience and chemical intuition, the assignment of such disconnected electron densities is often far from obvious. In this study, we suggest the use of molecular dynamics (MD) simulations and free energy calculations, which are well-established computational methods, to aid in the assignment of ambiguous disconnected electron densities. Specifically, estimates of (i) relative binding affinities, for instance between an ion and water, (ii) absolute binding free energies, i.e., free energies for transferring a solute from bulk solvent to a binding site, and (iii) stability assessments during equilibrium simulations may reveal the most plausible assignments. We illustrate this strategy using the crystal structure of the fluoride specific channel (Fluc), which contains five disconnected electron densities previously interpreted as four fluoride and one sodium ion. The simulations support the assignment of the sodium ion. In contrast, calculations of relative and absolute binding free energies as well as stability assessments during free MD simulations suggest that four of the densities represent water molecules instead of fluoride. The assignment of water is compatible with the loss of these densities in the non-conductive F82I/F85I mutant of Fluc. We critically discuss the role of the ion force fields for the calculations presented here. Overall, these findings indicate that MD simulations and free energy calculations are helpful tools for modeling water and ions into crystallographic density maps.
Highlights
130000 macromolecular structures have been deposited in the Protein Data Bank (PDB) by 2017 [1, 2], of which more than 120000 have been determined using X-ray crystallography or cryo-electron microscopy
We carried out 30 replicas with fluoride starting at F82 and F85, among which in 10 replicas (80 ns each) fluoride specific channel (Fluc) was blocked by L2 monobodies (Fig 1A), while in 20 replicas (40 ns each) Fluc was free of monobodies (Fig 1C)
The present study suggests that free-energy calculations and free molecular dynamics (MD) simulations may aid the assignment of disconnected electron densities in the crystal structure of proteins
Summary
130000 macromolecular structures have been deposited in the Protein Data Bank (PDB) by 2017 [1, 2], of which more than 120000 have been determined using X-ray crystallography or cryo-electron microscopy. Malde and Mark (2011) [27] showed that MD simulations and free energy calculations provide a useful framework to assign ligands in X-ray crystal complexes This proved to be a succesful strategy for determining the stereochemistry of the HIV-1 protease inhibitor JG-365 (PDB ID 7HVP) [28, 29] or the orientation of the ligand L-Serine in the binding pocket of Pyrococcus abyssi threonil-tRNA synthetase (PDB ID 2HKZ) [30]. Because ions and water play key functional roles in many proteins, the correct assignment of such densities may aid the understanding of protein function at molecular level With this aim in mind, we here investigate electron densities of the fluoride extruding Fluc channel. For the four densities at the F82 and F85 residues, free-energy calculations as well as stability assessments strongly favor water over fluoride
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