Macromolecular Crystallography for Synthetic Abiological Molecules: Combining xMDFF and PHENIX for Structure Determination of Cyanostar Macrocycles

Abstract

Crystal structure determination has long provided insight into structure and bonding of small molecules. When those same small molecules are designed to come together in multi-molecular assemblies, such as in coordination cages, supramolecular architectures and organic-based frameworks, their crystallographic characteristics closely resemble biological macromolecules. This resemblance suggests that bio-macromolecular refinement approaches be used for structure determination of abiological molecular complexes that arise in an aggregate state. Following this suggestion we investigated the crystal structure of a pentagonal macrocycle, cyanostar, by means of biological structure analysis methods and compared results to traditional small molecule methods. Cyanostar presents difficulties seen in supramolecular crystallography including whole molecule disorder and highly flexible solvent molecules sitting in macrocyclic and intermolecule void spaces. We used the force-field assisted refinement method, molecular dynamics flexible fitting algorithm for X-ray crystallography (xMDFF), along with tools from the macromolecular structure determination suite PHENIX. We found that a standard implementation of PHENIX, namely one without xMDFF, either fails to produce a solution by molecular replacement alone or produces an inaccurate structure when using generic geometry restraints, even at a very high diffraction data resolution of 0.84 Å. The problems disappear when taking advantage of xMDFF, which applies an optimized force field to re-align molecular models during phasing by providing accurate restraints. The structure determination for this model system shows excellent agreement with the small-molecule methods. Therefore, the joint xMDFF-PHENIX refinement protocol provides a new strategy that uses macromolecule methods for structure determination of small molecules and their assemblies.

Crystal Structure and Conformational Change Mechanism of a Bacterial Nramp-Family Divalent Metal Transporter

“…We used a ScaNramp-based homology model and the Fab fragment crystal structure as molecular replacement search models, with additional phasing provided by single-wavelength anomalous signal from three osmium ions bound to the Fab or at crystal contacts (Figure S1). Refinement was facilitated by using xMDFF (McGreevy et al, 2014; Singharoy et al, 2015). In particular, we used a combination of xMDFF and steered molecular dynamics to optimize our TM1a model, exploring different TM1a positional registries corresponding to a screw axis rotation of approximately one helical turn.…”

Inchworm movement of two rings switching onto a thread by biased Brownian diffusion represent a three-body problem

“…A second example involved threading of two planar cyanostar (CS, Fig. 1A) macrocycles onto a tetrazine thread (12,(33)(34)(35)(36)(37). The threading mechanism was not determined (12) because CV signatures did not conform to the expected production of an intermediate, as observed with copper(I)-phenanthroline.…”

Advances in the molecular dynamics flexible fitting method for cryo-EM modeling

“…In this case xMDFF was used to resolve uncertainty in the low-resolution (3.6, 4, and 7 Å) data in regard to the placement of the important S4 helix involved in the conformational change required for the function of the protein. xMDFF has also been extended to small molecule crystallography, a field not typically explored by macromolecular crystallography programs [80]. Nevertheless, when small molecules come together in multi-molecular assemblies, their structural characteristics closely resemble biomolecules and, thus, can greatly benefit from macromolecular refinement techniques such as xMDFF.…”

“…A small abiological molecule, cyanostar, exhibited whole molecule disorder and pushed the limits of small-molecule crystallography. However, a hybrid xMDFF-PHENIX approach was able to successfully refine the cyanostar structure and identify multiple conformations contained within the crystal [80]. Importantly, a key contribution to the success of the project was the development of accurate force field parameters using the force field toolkit (ffTK) plugin in VMD [81].…”