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abstracts
Membrane proteins and large assemblies are currently a major focus of molecular biology and molecular medicine. Due to their size and flexibility, these structures may only yield poor quality crystals for which diffraction intensities can be measured to merely mid-low resolution. Nevertheless, these data contain valuable structural information. Here, it will be shown how new features in COOT [1], REFMAC5 [2] and ProSMART [3] can help to exploit low resolution data for model building and refinement, as well as aid model validation. Refinement at low resolution can be stabilised with regularisers, such as jelly-body and external restraints. These allow to routinely obtain good quality models even in cases where only low-resolution data are available (e.g. >3Å). External restraints (available for protein and DNA/RNA) exploit structural prior knowledge, utilising the assertion that local interatomic distances should agree with previous observations. Sources for such prior knowledge include isomorphous and homologous structures, hydrogen bonding patterns, and typical conformations of secondary structure elements. Importantly, global rigidity is not enforced by these restraints - the approach presented allows for dramatic conformational differences between target and reference models. Consequently, restraints may be generated using homologous reference models resolved in different crystal forms. COOT facilitates model building at low resolution by removing degrees of freedom through so-called "backrub rotamers" and torsion angle restraints, as well as providing semi-automatic building options such as model morphing and jiggle fit . Map sharpening and blurring, now available in both COOT and REFMAC5, can be employed to provide further insight regarding the validity of a model, as well as aiding the model building process. General guidelines for the application of these features are provided, along with examples demonstrating their usage.
abstracts
Structural comparison often aids insight into the function and mechanics of biological macromolecules. To make such analyses more accessible, we present the Procrustes Structural Matching Alignment and Restraints Tool (ProSMART), which is designed to allow fast but detailed comparative analysis of macromolecular structures despite potential dissimilarities in global arrangement, such as domain motion and distortion. Whilst obtaining a residue alignment between structures is a prerequisite for comparative analysis, conventional alignment methods may fail in cases where conformational differences are dramatic. However, ProSMART achieves a conformation-independent structural alignment by focusing purely on local dissimilarities, rather than enforcing chain/domain rigidity. This allows the sensible comparison of protein (or DNA/RNA) structures in the presence of conformational change. ProSMART allows analysis of the structural conservation of local backbone and side chains in a wide variety of scenarios - the method is sensitive enough to allow identification of subtle dissimilarities between structures sharing high sequence homology, whilst being versatile enough to allow identification of local similarities between more distantly-related structures. In addition, ProSMART can be used for the identification of conserved rigid substructures, which may or may not represent functional domains. ProSMART is also used for the generation of external restraints for use in crystallographic refinement. Results from ProSMART can be visualised in either CCP4mg or PyMOL. All residue-based scores are illustrated using intuitive colour gradients, allowing easy visual assessment of local backbone and side chain conservation. Complementary structural comparison tools such as ProSMART can help break the complexity of the constantly growing pool of available structural data into a more readily accessible form, and consequently may aid biological insight into macromolecular structures.
