CCP4 Grant Renewal 2014-2019: Question-driven crystallographic data collection and advanced structure solution

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This proposal incorporates five related work packages.

In WP1 we will track synchrotron-collected data through computational structure determination, to find whether the most useful data can be recognised a priori using established or novel metrics of data quality and consistency. We will then enable data collection software to communicate with pipelines and graphics programs to assess when sufficient data have been collected for a given scientific question, and so to prioritise further beamtime usage. We will also communicate extra information about diffraction data to structure determination programs, and so support the statistical models and algorithms being developed in WP4.

WP2 will improve the key MR step of model preparation, especially from diverged, NMR, or ab initio models. One development will be to extend the size limit of ab initio search model generation by exploiting sequence covariance algorithms.

In WP3 we will use our description of electron density maps as a field of control points to better use electron density or atomic models positioned by MR. Restrained manipulation of these points provides a low-order parameterisation of refinement decoupled from atomic models, and therefore suitable for highly diverged atomic models or EM-derived maps. We will extend this approach to characterise local protein mobility without the requirement of TLS for predefinition of rigid groups.

In WP4 we will statistically model non-idealities in experimental data, including non isomorphism, spot overlap, and radiation damage. The resulting models, implemented in REFMAC, will be applied to refinement using data that are annotated by WP1 tools and tracked by WP0.

WP0 will provide the tools to integrate the other WPs. For this, it will create a cloud environment where storage- and compute-resources can be utilised optimally, and where rich information can be passed among beamlines, pipelines, and graphics programs.

The generic importance of macromolecular crystallography in general and CCP4 in particular is provided in the Pathways to Impacts section.

Molecular Replacement (MR) is an increasingly common route to solving the phase problem for protein crystal structures, its popularity arising from being fast and cheap. In 2012, 77% of protein structures submitted to the PDB were tagged as solved using MR. MR ranges in difficulty from positioning models that accurately model the scattering from the entire asymmetric unit to positioning very small components of the total scattering with high error. Essential to the success of MR is the availability of a search model that represents a portion of the unknown structure accurately enough that, once placed, it provides approximate phasing information allowing for further interpretation of the resulting electron density maps. WP2 aims to improve the efficiency and applicability of MR, enhancements that will reduce the time spent by the crystallographer on structure solution and extend the proportion of targets soluble by the technique. To do this WP2 will improve methods to assemble search models from conventional sources such as homology models and NMR structures. It will further build on recent innovations exploiting a novel source of structural information - low computational-cost, fragment-assembly derived ab initio models, as implemented in the CCP4 program AMPLE, extending the method to membrane proteins. The nascent technique of predicted contact-based ab initio modelling will further be explored, potentially allowing some large novel protein folds to be solved by MR for the first time. The predominance of MR as a structure solution method ensures that the entire crystallographic community will benefit from these improvements and so, in turn, will researchers in the many biological communities for whom protein structure information is valuable.

The software developed in WP2 will be added to the CCP4 suite. The CCP4 suite is used world-wide and is available on Windows, Linux and Mac_OS platforms, providing a direct distribution channel to macromolecular crystallographers. CCP4 has recently introduced and automated update mechanism to enable faster access to new developments. As a result developments in WP2 will be available immediately to the user community.

Although the focus in WP2 is on structural bioinformatics for crystallographic ends, we envisage that some of the methods we will develop to process and refine ab initio models will prove valuable to a broader bioinformatics community. For example, refinement of predicted contact-based models with Rosetta or other fragment-based protocols has not yet been done. Our benchmarking will indicate whether it provides a general method to improve local or global quality of the models: such a protocol would obviously be valuable to a broad modelling community. Similarly, incorporation of predicted contacts from the latest generation of covariance software into fragment assembly ab initio modelling is novel: the benefits of using larger or smaller numbers of predictions will become apparent through our benchmarking and will again be of broad benefit to protein modellers.