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CCP4 Advanced integrated approaches to macromolecular structure determination

ReferenceBB/S007040/1
Principal Investigator / Supervisor Dr Evgeny Krissinel
Co-Investigators /
Co-Supervisors		 Dr David Waterman, Dr Martyn Winn
Institution STFC - Laboratories
DepartmentScientific Computing Department
Funding typeResearch
Value (£) 275,180
StatusCurrent
TypeResearch Grant
Start date 01/07/2019
End date 30/06/2024
Duration60 months

Abstract

This proposal incorporates four related work packages. In WP1 we will expand on our work using established and novel metrics of data quality and consistency to quantify the relationship between diffraction and map quality. The tools will be used to optimise approaches to structure determination from multiple or serial crystallography data to enable optimal selection of collected data and fully utilise all the information in structural refinement. WP1 will also develop and implement methods for electron diffraction data collection, integration and refinement. WP2 will utilise generalise the use shift field refinement and extend its usage to hybrid refinement approaches and develop new software libraries to enhance and speed up protein structure model building and refinement across a wide resolution range. In WP3 we will develop and implement the use of contact prediction methods for use in crystallography. It will help identify protein domain boundaries, define new search model approaches. The contact prediction approach will also be used to validate Molecular replacement solutions and assist in the interpretation of crystallographically derived protein:protein contacts. In WP4 we will develop a model for electron scatter from macromolecular samples to enable software development and experimental design. These models will be used to develop and implement new scaling algorithms for electron diffraction data within DIALS.

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 and electron diffraction 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 but also extend to use of electron microscopes which have gained much recent publicity with the Nobel prize being awarded to researchers from this field. 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. 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. Electron diffraction (ED) on 3D crystals represents a promising new area for macromolecular structural biology. The strength of the interaction means that the technique is suited to very small samples (nanocrystals), from which many rotation images can be collected before radiation damage limits diffraction. This makes the method extremely competitive with XFEL serial crystallography, with costs being orders of magnitude lower. Structural information from ED is complementary to that from X-ray diffraction, with ED able to locate hydrogen positions and assess the charged state of residues and ions in the sample. One of main factors preventing routine high quality structure determination by ED is difficulty in the interpretation of experimental data due to dynamic diffraction effects (multiple scattering of electrons). Currently, methods to model dynamic diffraction require a complete structural model and a high level of expertise to use. As a result, ED for macromolecules currently remains limited to use in extra-thin samples, where the dynamic diffraction component may be neglected. Successful accomplishment of WP4 will have the following impacts: 1) An efficient tool for the mathematical modelling of ED experiments will be created. This will further the understanding of effects relevant to ED experiments, leading to optimization of experimental protocols and choice of sample crystals 2) The manifestation of dynamic diffraction will be studied in detail sufficient to understand the limits of the ED technique in terms of crystal size, symmetry, plus the effect of mosaicity and disorder, which has a mitigating effect on multiple scattering. 3) This understanding will inform procedures for extrapolating ED intensities obtained from multiple crystals to the kinematic approximation limit. In this regime, existing MX data processing software may be used for scaling and merging of ED images. This would enable more regular handling of ED data through phasing and model building with established software packages for MX, such as CCP4. 4) An immediate practical impact will be in the development of "ED scaling" algorithms within the dials.scale module of the DIALS data-processing software, developed jointly by Diamond Ltd., CCP4 and the Lawrence Berkeley National Laboratory in California. This will allow researchers to process ED data using familiar tools from X-ray diffraction approach.
Committee Research Committee D (Molecules, cells and industrial biotechnology)
Research TopicsStructural Biology, Technology and Methods Development
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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