Automated de novo building of protein models into electron microscopy maps

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

The aim of the project is to adapt and optimize X-ray crystallographic model building methods for effective application to the de novo building of high resolution cryo-electron microscopy maps, and to distribute the software to broad user community. We will achieve these results by following the same approach to software development which we have successfully applied to previous projects:

- Firstly, a curated library of test data will be prepared for which the final structures are known. The use of test data in this way has proven critical in our previous work, as it allows every change to the software to be evaluated across a representative selection of datasets, avoiding the problem of software which only works on a single structure. It is envisaged that a full set of tests will be run on a daily to weekly time scale, depending on the time requirements. If necessary more frequent tests will be run on a subset of the data.

- The software will be developed in the CCP4 source code repository, providing a full history of changes to the software. The current version of the software will be recorded against each set of test results to document the effectiveness of each change to the code. The source code repository is publicly visible.

- The methods used will be those developed as part of the BUCCANEER, NAUTILUS and COOT software packages, modified to provide best results against the test data.

- Release versions of the software will be incorporated into the CCP4 and CCP-EM build frameworks to allow the software to be built for Windows, Mac and Linux computer systems.

- The software will be incorporated into automate software pipelines and presented to the user through the standard CCP4 and CCP-EM user interfaces.

- The software will be incorporated into the standard CCP4 and CCP-EM installation and update tools to allow installation by normal users without the assistance of a system manager.

Cryo-electron microscopy has progressed over the last decade from being a niche technique for the low resolution imaging of large complexes, to a comparatively routine technique suitable for solving most medium to large structures. The resolution of the best EM reconstructions are now sufficient for de novo building. The UK now boasts a number of large EM facilities, for example at the MRC, Diamond and Leeds.

While the capital cost of an EM facility is large, the method offers substantial benefits for certain classes of problem. Unlike X-ray crystallography, the sample does not need to be crystallized - a time consuming and sometimes unsuccessful step. The challenge of crystallisation introduces a risk in the crystallographic pathway which carries its own cost. Consequently EM will see increasing use in the biotech and pharmaceutical industries. EM methods have the further benefit of imaging molecules in a state which is undistorted by crystal contacts, and thus in some cases more informative for biological problems.

CCP4 has been very successful in serving the biotech and pharmaceutical industries, as evidenced by over a hundred annual software licenses issued to industrial customers, raising typically £1m per annum in income. CCP-EM seeks to fill the same role for EM users. Dr Cowtan's work has contributed significantly to the success of CCP4, with his contributions to density modification, model building, visualisation and supporting infrastructure attracting over 10,000 citations in the peer-reviewed literature, as well as being cited in patents.

The development of de novo model building software specialised to the interpretation of EM maps, and their contribution to the CCP-EM software suite for EM structure solution will make the method more effective and make de novo structure solution more accessible to the typical user. The validation and automated rebuilding of existing models will reduce bias and improve the quality of the structures in the EM database.

The direct benefits to industry are expected, by parallel with X-ray crystallography, to be realised through the development of new drugs and biochemical processes, building on the insights arising from the structures determined by these methods. However, as with X-ray crystallography, we expect that linking individual products to software developments will be difficult due to the closed nature of the sector. The primary indicator of impact will remain the license fees which industrial users are willing to pay for the software.

Finally, the theoretical work which underlies this proposal will improve our understanding of the features of EM electron density reconstructions, and provide a basis for other developers to address the same problems in different ways. We therefore expect that our work will provide a catalyst for an expansion of the development of software for the later stages of EM structure solution, in particular model building and refinement. UK leadership in this area will provide a competitive advantage to our users and partners in UK industry.