High-throughput low-volume crystallisation facility

Lead Research Organisation: University of Leeds
Department Name: Institute of Membrane & Systems Biology

Abstract

This is an equipment proposal, to enable ground-breaking research. Almost all of our understanding of how proteins and nucleic acids such as DNA work has come from structural biology. This requires growing crystals of these macromolecules, as in the pioneering work of Max Perutz, who won a Nobel prize in 1962 for solving the structure of the oxygen-carrying protein of the blood, haemoglobin. The hardest things to crystallise are the proteins that sit in the membranes that surround living cells, because they are not soluble in water - but these proteins are the targets for 50% of all drugs. They are also the ones that turn sunlight into energy, conduct nerve impulses and transport nutrients of all kinds into cells.

Over the last ten years, there has been a revolution in methods for crystallising proteins, especially membrane proteins, and our proposal is to equip the University of Leeds, and thus other local universities (Sheffield, Huddersfield, Newcastle and Manchester) with this cutting-edge equipment. The equipment has three components: (1) characterisation equipment (SEC-MALLS, LCP-FRAP), which will help us determine if the protein is likely to be crystallisable in the conditions being used; (2) a crystallisation robot so that we can use 20-50 times less protein than before ("drops" of 20-50 nl, rather than 1 ul) in each crystallisation trial; and (3) robotic imagers both at 4 C and room temperature. As we will be doing tens of thousands of trials, robotic imagers make visualising the experiments much easier than having to look at each experiment one by one under a microscope. In addition, the crystallisation robot can make "lipidic cubic phase" (LCP) drops, which corresponds to squeezing out 50 nl of toothpaste at a time. LCP has in particular revolutionised the crystallisation of membrane proteins but, like toothpaste, it is opaque. Consequently, we are also buying a "SONICC" imager, which will enable us to see very small protein crystals in the opaque LCP.

The post-genomic era has provided unimagined insights into the chemistry and regulatory mechanisms underlying life, and structural biology has been an very important part of this. Despite successes with water-soluble proteins, major challenges remain, particularly for membrane proteins and large mammalian/eukaryotic protein complexes, which this equipment will address. The structural work at the Astbury Centre for Structural Molecular Biology is centred around four major overlapping theme areas: (1) Membrane proteins; (2) large complexes; (3) pathogen-host interactions; and (4) design of small molecules (i.e. drugs).

Examples of projects where we expect breakthroughs are: how do the ion-channels involved in sensing pain, temperature or taste work? How do viruses that contain RNA, like the common cold or smallpox, package the RNA inside themselves? This is required for the virus to be infective. How do large molecular machines, like the vacuolar ATPase, work and how are they regulated? These are important in trypanosomal parasites that cause major diseases in both animals and humans. Can we understand better how some plants resist the toxicity of metals such as aluminium, and can we therefore enhance this ability in major crops? This will help make crops grow better, with less use of fertilisers, in acidic soils. Finally, bacteria that accumulate on surfaces form biofilms - around teeth, around prosthetic implants, on the surfaces of ships, with adverse consequences. Understanding how this happens and preventing it requires understanding the structures of the proteins involved.

Technical Summary

Our goal is to add a modern crystallisation section to our structural pipeline. We lack the critical equipment components.
The main technical objectives are thus to:

(1) Acquire a size-exclusion chromatography setup with multiangle laser light scattering (MALLS), refractive index and dynamic light scattering detectors. With this, we wil be able to determine parameters critical for crystallisation such as: the absolute molecular mass of the peak on the column as a function of time, the protein:detergent ratio in the peak, and if it is monodisperse or not.
(2) Acquire crystallisation robotics including lipidic cubic phase technology (LCP), which has become an essential component of membrane protein crystallography.
(3) Acquire robotic imaging at 20 and 4 C, so that we can routinely scan 96-well crystallisation plates and deliver those images to internal and external users. This should include a SONICC-TPEF imager, which allows the detection of very small (micron-sized) protein crystals in the opaque LCP matrix.
(4) Acquire a lipidic cubic phase-fluorescence recovery after photobleaching (LCP-FRAP) setup to determine the mobility of proteins in the LCP matrix. Proteins that are not mobile do not crystallise.

These new tools, which form a consistent whole that exists nowhere in the North of England, will eliminate the major bottleneck in our current structure solution pipeline. The improvements will thus both enhance the productivity of current users and increase the number of in-house and external users.

Our scientific goals will be to use the new equipment to characterise and crystallise a variety of high-value targets, including TRP and Kv channels, vacuolar ATPases and pyrophosphatases, nucleoside transporters and RNA-virus protein complexes.

Our milestones will be purchase, delivery and installation, use by internal users, and use by external users. We will have a workshop to introduce users to the new equipment and techniques.

Planned Impact

This is an equipment grant for enabling technology to complete the structural biology pipeline at the University of Leeds from protein production to x-ray diffraction and structure solution by providing the vital missing intermediate step: modern characterisation, crystallization and imaging equipment. The academic impact is thus on solving breakthrough structures. Who are the other beneficiaries?

SMEs, big pharmaceutical and agribusiness companies will benefit from this research. We already work with a number of such companies, including MedImmune, GlaxoSmithKline, AstraZeneca and Aptamer Solutions, and structures of important agricultural or pharmaceutical targets will be of direct benefit to them as they will provide the basis for design of new small molecules (e.g. antibacterials, antifungals). The research will also lead to better understanding of how proteins work, which is of great importance to SMEs in synthetic biology and in metabolic engineering. We have in the past and will in the future patent our discoveries, so the work will lead to improving the economic competitiveness of the EU in general and the UK in particular. The timeframe for drug development is 5-15 years.

A number of the structures are of relevance for public health and animal welfare, and so for international policy and policy makers. This is particularly true for those like the vacuolar ATPase and pyrophosphatase, which are important in trypanosomal diseases such as sleeping sickness. Trypanosomiasis reduces human and animal productivity across large swathes of sub-Saharan Africa. New approaches and drugs will thus improve the quality of life in the developing world in particular. Some of the work has benefits for government agencies and regulators because it will lead to new ways of determining the effects of food additives and drugs: the effects can be studied on the target molecules, rather than via animal testing. The timeframe for this is the next 5-10 years.

There are three immediate benefits to this proposal. First, the immediate research environment in the North of England benefits hugely, as the complete structural biology pipeline proposed does not exist here. It thus meets the goals of the research councils to coordinate and minimize duplication, and it provides important regional support so that Leeds and the North maintain their traditional strength in structural biology, rather than have it all concentrated within 80 miles of Whitehall.

Second, all of the investigators, and the University of Leeds, are committed to outreach to the local and UK community in a variety of ways: inviting schoolchildren in to the University, having them do summer projects in laboratories, giving talks and radio interviews, and acting as a consultants for outreach efforts aimed at teenagers.

Finally, a major transferrable benefit of all academic research is the people trained during the project. The scientists using the equipment acquired through this grant will acquire professional skills that they can use in research-based biotechnological industry. In addition the University of Leeds has an extensive career development program that will provide transferrable skills. These trained people as they move to other institutions in academia, in government and in industry, will affect the larger society positively in all the ways described above.

Publications

10 25 50

 
Description As mentioned under Impact, it is strange to report on the activities of about 30 other laboratories. The grant was for enabling technology, not research. That being said, we can say that we and other groups have used the infrastructure to crystallise new proteins and obtain novel protein structures. Four examples are as follows:
1) The infrastructure underpins x-ray crystallisation and structural biology in the Astbury Centre for Structural and Molecular Biology. As such, it is used by a great number of different groups, and enables graduate students and PDRAs to make progress in their research, as evidence by the papers published. These papers include ones in Plant Cell and Nature Communications.
2) The infrastructure has been instrumental in creating new collaborations, including underpinning the Leeds involvement in two EU Innovative Training Networks, RAMP (funded in 2017) and ViBrANT (funded in 2018). The former is focussed specifically on developing new technologies to crystallise membrane proteins, which the crystallisation robots bought as part of the grant are ideal for. The consortium involves two of the original PIs (Goldman and Pearson); Pearson has now moved to the University of Hamburg, as well as leading players in membrane protein structural biology from inter alia France, Ireland, Sweden, Denmark and two major pharmaceutical companies (Novartis and AstraZeneca) as well as three SMEs. This is a major new research network.
3) New structures of pyrophosphatases that have the potential to lead to drug design against malaria parasites, and published in Nature Communications in 2016; new potential inhibitors have already been designed, including ones that kill malaria parasites at micromolar concentrations.
4) New work has led to extending the molecular recognition achieved by enzymes (work published in PNAS in 2017). this will allow better enzyme design and redesign.
5) New work on binding of adhirons to Fc receptors led to understanding how to bind specific FcRs for therapeutic design (work published in PNAS in 2018).
6) The work has led to new models of how the aurora kinase work: this is important in various cancers.
Exploitation Route The research findings have been in the areas of Adhirons, and how they bind to various molecules. These are being developed as potential diagnostic tools and in principle for clinical use. Structures of viral proteins and of proteins from malaria parasites are being used for drug design. Work on proteomimetics may lead to novel protein-like structures that will have the functions of enzymes but without some of the drawbacks (for instance, improved (thermo)stability; not being degraded in the stomach and so on).
Sectors Agriculture, Food and Drink,Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The grant was for enabling technology for all of the structural biology laboratories at the University of Leeds; and the way the question is phrased implies that I should be able to tell what 30 other laboratories have done - not only their research, but further developments. I think we can assume that, from the published papers, the research has been useful in identifying new approaches to drugs and to biotechnology: the standard immediate outputs for protein structures. Another major impact, of course, has been that students have used the infrastructure, and it has helped them graduate and achieve their PhDs. All grants have this as an output.
First Year Of Impact 2016
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Confidence in Concept Award
Amount £65,750 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 03/2018 
End 02/2020
 
Description David Phillips Fellowship
Amount £1,274,037 (GBP)
Funding ID BB/N019970/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2016 
End 10/2021
 
Description Fellowship for Julie Heggelund
Amount £321,000 (GBP)
Organisation Research Council of Norway 
Sector Public
Country Norway
Start 01/2017 
End 12/2019
 
Description Marie Curie Innovative Training Network
Amount € 3,243,300 (EUR)
Organisation European Union 
Sector Public
Country European Union (EU)
Start 04/2017 
End 03/2021
 
Description Marie Curie Innovative Training Network (ViBrANT)
Amount € 4,500,000 (EUR)
Funding ID 765042 
Organisation European Union 
Sector Public
Country European Union (EU)
Start 01/2018 
End 12/2022
 
Description Marie Curie postdoctoral fellowship
Amount € 183,000 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 09/2016 
End 08/2018
 
Description Marie Curie postdoctoral fellowship
Amount € 183,000 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 09/2016 
End 08/2018
 
Description Marie Curie postdoctoral fellowship
Amount € 183,000 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 07/2015 
End 06/2017
 
Description Responsive mode call
Amount € 608,000 (EUR)
Organisation Academy of Finland 
Sector Public
Country Finland
Start 09/2015 
End 08/2019
 
Description University of Leeds graduate student fellowship scheme
Amount £45,000 (GBP)
Organisation University of Leeds 
Sector Academic/University
Country United Kingdom
Start 08/2014 
End 07/2017
 
Description Wellcome Trust ISSF
Amount £50,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2018 
End 08/2019
 
Description White Rose graduate fellowship scheme
Amount £45,000 (GBP)
Organisation White Rose University Consortium 
Sector Academic/University
Country United Kingdom
Start 08/2015 
End 07/2018
 
Title PDB CODES 
Description These are structures generated by groups who have used the crystallisation robots. The PDB codes are: 5C3F, 5C3G, 5A36, 5A37, 5A38, 5A4B, 5IU1, 5A0O, 5C3F, 5C3G 5A8G, 5A97 5AG2, 4X9Q 4CRZ, 4CS0, 5A0O, 6ELK, 5ELJ, 5EQL, 5ELU, 5LS7, 6EZZ, 6GRR, 6FHK, 5Y17, 5XVZ, 5XY4, 5ZZ1, 6F9I, 6EJN, 5ODS, 5ODT, 5MN2 and 5ML9 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact not applicable 
URL http://www.ebi.ac.uk/pdbe/
 
Description Ace1 transport proteins 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution provide intellectual input on protein structure solution; advise Marie Curie Fellow
Collaborator Contribution Provide mechanistic studies on the Acinetobacter baumannii Ace1 protein, provide protein for crystallography
Impact Crystallography, allied with protein production, membrane biology, biophysical and biochemical studies
Start Year 2016
 
Description Collaboration on Fc gamma Receptors 
Organisation University of Leeds
Department University of Leeds Special Collections
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been responsible for solving the structures of a series of Fcgamma receptors, both mutant and wild-type, examining their complexes, performing SEC-MALLs analysis, and analysing what the structures mean for the interactions of Fcs with their receptors
Collaborator Contribution Our partners have been involved in identifying intriguing Fcgamma receptor mutations using genome-wide association studies, in studying the effects of these receptors in cell-based assays, and in developing novel binding partners (adhirons) that can inhibit specific Fcgamma receptors.
Impact Robinson, J. I., Baxter, E. W., Owen, R. L., Thomsen, M., Tomlinson, D. C., Waterhouse, M. P., Win, S. J., Nettleship, J. E., Tiede, C., Foster, R. J., Owens, R. J., Fishwick, C. W. G., Harris, S. A., Goldman, A., McPherson, M. J. & Morgan, A. W. Affimer proteins inhibit immune complex binding to Fc?RIIIa with high specificity through competitive and allosteric modes of action. Proc. Natl. Acad. Sci. U S A 115, E72-E81 (2018).
Start Year 2016
 
Description Collaboration on TAAs 
Organisation University of Frankfurt
Country Germany 
Sector Academic/University 
PI Contribution This is part of the ViBrANT Innovative training network on bacterial and viral adhesion proteins. Our role in this collaboration is to solve the structures of different trimeric autotransporter adhesins (TAAs) bound to potential ligands, as the basis for interfering with their function.
Collaborator Contribution The partners are involved in producing the TAAs, and in studying their biological and biophysical properties.
Impact none yet
Start Year 2017
 
Description Collaboration on TAAs 
Organisation University of Oslo
Country Norway 
Sector Academic/University 
PI Contribution This is part of the ViBrANT Innovative training network on bacterial and viral adhesion proteins. Our role in this collaboration is to solve the structures of different trimeric autotransporter adhesins (TAAs) bound to potential ligands, as the basis for interfering with their function.
Collaborator Contribution The partners are involved in producing the TAAs, and in studying their biological and biophysical properties.
Impact none yet
Start Year 2017
 
Description Collaboration on structure of Kv channels 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Purification of the Kv1.2 channel from Pichia pastoris for crystallisation
Collaborator Contribution Reagents for the project; clones; advice
Impact Collaboration is not multidisciplinary. Outcomes: purified protein for crystallisation
Start Year 2013
 
Description Collaboration on the Comatose ABC family D transporter 
Organisation Rothamsted Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Setting up for crystallisation of the comatose protein
Collaborator Contribution Production of the comatose protein
Impact none so far
Start Year 2014
 
Description Collaboration on the Comatose ABC family D transporter 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Setting up for crystallisation of the comatose protein
Collaborator Contribution Production of the comatose protein
Impact none so far
Start Year 2014
 
Description Collaboration with Nankai 
Organisation Nankai University
Country China 
Sector Academic/University 
PI Contribution Will provide clones and house a graduate student for the purpose of collaborative research
Collaborator Contribution Provides graduate student for two years to perform research on membrane proteins
Impact 10.1371/journal.pone.0143010
Start Year 2014
 
Description Structural studies of mutants of Mhp1 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Advice on crystallising the Mhp1 mutants; access to the crystallisation robotics
Collaborator Contribution production, purification, structure solution.
Impact Initial draft of a manuscript on the Mhp1 mutants
Start Year 2014
 
Description Participation in open day activities associated with the Astbury Conversation 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 300 members of the general public, including school-children were part of the open sessions at the Astbury conversation. The keynote speaker in 2018 was the Nobelist Brian Kobilka. There was increased interest in the idea of using structural understanding to develop much improved drugs - for instance in the opioid family of drugs.
Year(s) Of Engagement Activity 2018
 
Description School visits for crystallisation facility 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 30 pupils visited the BBSRC crystallisation facility as part of open days. It led to increased interest and enthusiasm for structural biology.
Year(s) Of Engagement Activity 2014,2015
 
Description Student visits for the Baldwin memorial symposium 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 127 keystone 4 pupils from local schools attended a public lecture by the Nobelist Professor Sir John Walker, FRS, and also were involved in a visit to the laboratories. They had an introduction to scientific techniques, sparking interest in biological research areas.
Year(s) Of Engagement Activity 2015
URL http://www.fbs.leeds.ac.uk/baldwin/