Super-resolution biomolecular crystallography with low-resolution data

Abstract

X-ray crystallography has become the most common method used by structural biologists to obtain three-dimensional structures of proteins and protein–protein complexes. However, crystals of large macromolecular complexes often diffract only weakly — yielding a resolution of less than about 4 Å — so it is important to develop new methods that work at such low resolution. Here, the authors show that information from comparative modelling can be combined in a statistically controlled fashion with the observed diffraction data in order to achieve a structure from low-resolution diffraction data that has a similar quality as a high-resolution structure. X-ray crystallography has become the most common way for structural biologists to obtain the three-dimensional structures of proteins and protein complexes. However, crystals of large macromolecular complexes often diffract only weakly (yielding a resolution worse than 4 Å), so new methods that work at such low resolution are needed. Here a new method is described by which to obtain higher-quality electron density maps and more accurate molecular models of weakly diffracting crystals. X-ray diffraction plays a pivotal role in the understanding of biological systems by revealing atomic structures of proteins, nucleic acids and their complexes, with much recent interest in very large assemblies like the ribosome. As crystals of such large assemblies often diffract weakly (resolution worse than 4 Å), we need methods that work at such low resolution. In macromolecular assemblies, some of the components may be known at high resolution, whereas others are unknown: current refinement methods fail as they require a high-resolution starting structure for the entire complex1. Determining the structure of such complexes, which are often of key biological importance, should be possible in principle as the number of independent diffraction intensities at a resolution better than 5 Å generally exceeds the number of degrees of freedom. Here we introduce a method that adds specific information from known homologous structures but allows global and local deformations of these homology models. Our approach uses the observation that local protein structure tends to be conserved as sequence and function evolve. Cross-validation with Rfree (the free R-factor) determines the optimum deformation and influence of the homology model. For test cases at 3.5–5 Å resolution with known structures at high resolution, our method gives significant improvements over conventional refinement in the model as monitored by coordinate accuracy, the definition of secondary structure and the quality of electron density maps. For re-refinements of a representative set of 19 low-resolution crystal structures from the Protein Data Bank, we find similar improvements. Thus, a structure derived from low-resolution diffraction data can have quality similar to a high-resolution structure. Our method is applicable to the study of weakly diffracting crystals using X-ray micro-diffraction2 as well as data from new X-ray light sources3. Use of homology information is not restricted to X-ray crystallography and cryo-electron microscopy: as optical imaging advances to subnanometre resolution4,5, it can use similar tools.