Case, D. A. | Search bioRxiv

bioRxiv2019

Improved chemistry restraints for crystallographic refinement by integrating the Amber force field into Phenix

The refinement of biomolecular crystallographic models relies on geometric restraints to help address the paucity of experimental data typical in these experiments. Limitations in these restraints can degrade the quality of the resulting atomic models. Here we present an integration of the full all-atom Amber molecular dynamics force field into Phenix crystallographic refinement, which enables a more complete modeling of biomolecular chemistry. The advantages of the force field include a carefully derived set of torsion angle potentials, an extensive and flexible set of atom types, Lennard-Jones treatment of non-bonded interactions and a full treatment of crystalline electrostatics. The new combined method was tested against conventional geometry restraints for over twenty-two thousand protein structures. Structures refined with the new method show substantially improved model quality. On average, Ramachandran and rotamer scores are somewhat better; clash scores and MolProbity scores are significantly improved; and the modelling of electrostatics leads to structures that exhibit more, and more correct, hydrogen bonds than those refined with traditional geometry restraints. We find in general that model improvements are greatest at lower resolutions, prompting plans to add the Amber target function to real-space refinement for use in electron cryo-microscopy. This work opens the door to the future development of more advanced applications such as Amber-based ensemble refinement, quantum mechanical representation of active sites and improved geometric restraints for simulated annealing.\n\nIMPORTANTthis document contains embedded data - to preserve data integrity, please ensure where possible that the IUCr Word tools (available from http://journals.iucr.org/services/docxtemplate/) are installed when editing this document.\n\nSynopsisThe full Amber force field has been integrated into Phenix as an alternative refinement target. With a slight loss in speed, it achieves improved stereochemistry, fewer steric clashes and better hydrogen bonds.

molecular biology↗

bioRxiv2019

The interplay between molten globule intermediates and heme disassociation characterizes human hemoglobin disassembly

Hemoglobin functions as an oxygen transport protein, with each subunit containing a heme cofactor. We have developed a global disassembly model for human hemoglobin, linking hemin (ferric heme) disassociation and apo(heme-free)-protein unfolding pathways. The model was based on the evaluation of circular dichroism and visible absorbance measurements of guanidine hydrochloride-induced disassembly of holo (heme-bound)-hemoglobin and previous measurements of apohemoglobin unfolding. The populations of holo-intermediates and equilibrium disassembly parameters were determined quantitatively for adult and fetal hemoglobins. The key stages for disassembly into unfolded monomers are characterized by hemichrome intermediates with molten globule characteristics. Hemichromes, which occur when both hemin iron axial sites coordinate amino acids, are not energetically favored in native human hemoglobins. However, these hexacoordinate iron complexes are important for preventing hemin disassociation from partially unfolded species during early disassembly and late stage assembly events. Both our model evaluation and independent small angle X-ray scattering measurements demonstrate that heme disassociation during early disassembly leads to loss of tetrameric structural integrity. Dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells where the hemoglobin concentration is very high. This prediction explains why in the red cells of patients with unstable hemoglobinopathies, misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway is dominated by early hemin disassociation events, which leads to the generation of higher fractions of apo-subunits and free hemin known to damage to the integrity of blood vessel walls. Thus, our model illuminates the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis, and provides insights into the factors governing hemoglobin assembly during erythropoiesis.\n\nSignificanceOur deconvolution and global analysis of spectral data led to both the characterization of \"hidden\" hemichrome intermediates and the development of a quantitative model for human hemoglobin disassembly/assembly. The importance of this mechanism is several-fold. First, the hemoglobin system serves as a general biological model for understanding the role of oligomerization and cofactor binding in facilitating protein folding and assembly. Second, the fitted parameters provide: (a) estimates of hemin affinity for apoprotein states; (b) quantitative interpretations of the pathophysiology of hemoglobinopathies and other diseases associated with unstable globins and red cell lysis; (c) insights into the factors governing hemoglobin assembly during erythropoiesis; and (d) a framework for designing targeted hemoglobinopathy therapeutics.

biophysics↗