xMDFF: Flexible fitting for low-resolution X-ray Crystallography

Investigating the structure of large biomolecular complexes has posed a serious challenge to the traditional crystallography techniques. Inherent flexibility of such large systems, presence of disordered solvent and lipids or ligands often cause the crystals to diffract at low resolutions. Furthermore, in the low-resolution limit, the number of structural parameters to be determined from crystallography often exceeds that of the observed diffraction intensities. At moderate to low resolutions, knowledge of the stereochemistry of the system must be incorporated to achieve accurate atomic positions. The strengths of MDFF have been leveraged to address the difficulties of refining structures from low-resolution X-ray data to create xMDFF (MDFF for low-resolution X-ray Crystallography). For use with low-resolution X-ray crystallography, the MDFF protocol was modified to work with model-phased densities, which uses the phases calculated from a tentative model and the amplitudes from the X-ray diffraction data to produce a density map. Next, the tentative model is flexibly fitted into the electron density map using MDFF. The xMDFF-fitted structure provides new phases that, together with experimental amplitudes, are used to regenerate the electron density map. The fitted structure is then employed as an updated search model to be driven into the new model-phased density map, and this process continues iteratively.

xMDFF refinements have been shown to provide improved structures as characterized by multiple evaluators including lower Rwork and Rfree values, higher cross correlations, and improved structural geometry. xMDFF is capable of refining search models that must undergo large-scale deformations to reach the final structure and can handle flexible regions that often cause the low-resolution data.

Refinement of highly flexible region in case of 1XDV. Substantial density improvements are observed in a flexible region of 1XDV, illustrated by the difference in the density map between the initial (a) and xMDFF-refined final (b) structure; Local cross correlations increase from 0.47 to 0.63, implying a more unambiguous placement of the atoms.

ReMDFF: Flexible fitting for high-resolution cryo-electron Microscopy

Living cells are brimming with the activity of macromolecular complexes carrying out their assigned tasks. Structures of these complexes can be resolved with cryo-electron microscopy (cryo-EM), wherein the complexes are first freeze-shocked into states characterizing their action and subsequently imaged by detection cameras. Recent advances in direct detection camera technology enable today's cryo-EM laboratories to image the macromolecular complexes at high-resolution, giving us a better view of the cell than ever before. Computational techniques like molecular dynamics flexible fitting (MDFF) are a key tool for producing atomic models of the imaged molecules, providing greater insight into their structure and function. The increased resolution of EM maps, which contain sharp valleys capable of trapping structures, presents a challenge to MDFF which was originally developed for maps in a lower resolution range. However, our study unveils two new techniques called cascade (cMDFF) and resolution exchange (ReMDFF) molecular dynamics flexible fitting to overcome the hurdles posed by high-resolution maps. The refinement is achieved by interpreting a range of cryo-EM images, starting with an image of fuzzy resolution and progressively improving the image's contrast until near-atomic resolution is reached.