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

ReferenceBB/L009544/1
Principal Investigator / Supervisor Professor Daniel Rigden
Co-Investigators /
Co-Supervisors
Institution University of Liverpool
DepartmentInstitute of Integrative Biology
Funding typeResearch
Value (£) 39,443
StatusCompleted
TypeResearch Grant
Start date 30/06/2015
End date 29/06/2019
Duration48 months

Abstract

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.

Summary

Proteins, DNA and RNA are the active machines of the cells which make up living organisms, and are collectively known as macromolecules. They carry out all of the functions that sustain life, from metabolism through replication to the exchange of information between a cell and its environment. They are coded for by a 'blueprint' in the form of the DNA sequence in the genome, which describes how to make them as linear strings of building blocks. In order to function, however, most macromolecules fold into a precise 3D structure, which in turn depends primarily on the sequence of building blocks from which they are made. Knowledge of the molecule's 3D structure allows us both to understand its function, and to design chemicals to interfere with it. Due to advances in molecular biology, a number of projects, including the Human Genome Project, have led to the determination of the complete DNA sequences of many organisms, from which we can now read the linear blueprints for many macromolecules. As yet, however, the 3D structure cannot be predicted from knowledge of the sequence alone. One way to "see" macromolecules, and so to determine their 3D structure, involves initially crystallising the molecule under investigation, and subsequently imaging it with suitable radiation. Macromolecules are too small to see with normal light, and so a different approach is required. With an optical microscope we cannot see objects which are smaller than the wavelength of light, roughly 1 millionth of a metre: Atoms are about 1000 times smaller than this. However X-rays have a wavelength about the same as the size of the atoms. For this reason, in order to resolve the atomic detail of macromolecular structure, we image them with X-rays rather than with visible light. The process of imaging the structures of macromolecules that have been crystallised is known as X-ray crystallography. X-ray crystallography is like using a microscope to magnify objects that are too small to be seen with visible light. Unfortunately X-ray crystallography is complicated because, unlike a microscope, there is no lens system for X-rays and so additional information and complex computation are required to reconstruct the final image. This information may come from known protein structures using the Molecular Replacement (MR) method, or from other sources including Electron Microscopy (EM). Once the structure is known, it is easier to pinpoint how macromolecules contribute to the living cellular machinery. Pharmaceutical research uses this as the basis for designing drugs to turn the molecules on or off when required. Drugs are designed to interact with the target molecule to either block or promote the chemical processes which they perform within the body. Other applications include protein engineering and carbohydrate engineering. The aim of this project is to improve the key computational tools needed to extract a 3D structure from X-ray crystallography experiments. It will provide continuing support to a Collaborative Computing Project (CCP4 first established in 1979), which has become one of the leading sources of software for this task. The project will help efficient and effective use to be made of the synchrotrons that make the X-rays that are used in most crystallographic experiments. It will provide more powerful tools to allow users to exploit information from known protein structures when the match to the unknown structure is very poor. It will also automate the use of information from electron microscopy, even when the crystal structure has been distorted by the process of growing the protein crystal. Finally, it will allow structures to be solved, even when poor quality and very small crystals are obtained.

Impact Summary

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.
Committee Research Committee D (Molecules, cells and industrial biotechnology)
Research TopicsStructural Biology
Research PriorityX – Research Priority information not available
Research Initiative X - not in an Initiative
Funding SchemeX – not Funded via a specific Funding Scheme
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