Development of crystallographic methods for phasing highly modulated macromolecular structures

Abstract

Pathologies that result in highly modulated intensities in macromolecular crystal structures pose a challenge for structure solution. To address this issue two studies have been performed: a theoretical study of one of these pathologies, translational non- crystallographic symmetry (tNCS), and a practical study of paradigms of highly modulated macromolecular structures, coiled-coils. tNCS is a structural situation in which multiple, independent copies of a molecular assembly are found in similar orientations in the crystallographic asymmetric unit. Structure solution is problematic because the intensity modulations caused by tNCS cause the intensity distribution to differ from a Wilson distribution. If the tNCS is properly detected and characterized, expected intensity factors for each reflection that model the modulations observed in the data can be refined against a likelihood function to account for the statistical effects of tNCS. In this study, a curated database of 80482 protein structures from the PDB was analysed to investigate how tNCS manifests in the Patterson function. These studies informed the algorithm for detection of tNCS, which includes a method for detecting the tNCS order in any commensurate modulation. In the context of automated structure solution pipelines, the algorithm generates a ranked list of possible tNCS associations in the asymmetric unit, which can be explored to efficiently maximize the probability of structure solution. Coiled-coils are ubiquitous protein folding motifs present in a wide range of proteins that consist of two or more α-helices wrapped around each other to form a supercoil. Despite the apparent simplicity of their architecture, solution by molecular replacement is challenging due to the helical irregularities found in these domains, tendency to form fibers, large dimensions in their typically anisometric asymmetric units, low-resolution and anisotropic diffraction. In addition, the internal symmetry of the helices and their alignment in preferential directions gives rise to systematic overlap of Patterson vectors, a Patterson map that indicates tNCS is present, and intensity modulations similar to those in true tNCS. In this study, we have explored fragment phasing on a pool of 150 coiled-coils with ARCIMBOLDO_LITE, an ab initio phasing approach that combines fragment location with Phaser and density modification and autotracing with SHELXE. The results have been used to identify limits and bottlenecks in coiled-coil phasing that have been addressed in a specific mode for solving coiled-coils, allowing the solution of 95% of the test set and four previously unknown structures, and extending the resolution limit from 2.5 Å to 3.0 Å.