Centre for structural analysis of complex biological systems

Lead Research Organisation: University of Bristol
Department Name: Biochemistry


Understanding the function of the molecules of life requires knowledge of their three dimensional structures. Seeing, at or near to the level of individual atoms, how the building blocks of life (proteins and DNA) are assembled enables us to understand both how they may act to drive the chemical reactions that power and maintain living cells, and how they are organised into more complex structures that form the basis of cells and tissues. Detailed knowledge of structure can explain how specific alterations affect function, for example where changes to specific molecules are linked to disease, or how biological systems can be engineered to fulfil useful functions, such as making new drugs or turning carbon dioxide into liquid fuels.

Most structures of biological molecules are derived from experiments where ordered crystals of the pure material are exposed to X-rays. The success of this approach relies upon inducing crystals to form. Unfortunately, for many interesting and important biological molecules this remains very difficult, and large numbers of experiments must be conducted to identify suitable conditions for crystal formation. However, recent technological developments have increased the number of experiments possible with limited amounts of material, and created automated systems to monitor the progress of experiments and detect crystals as they form. Furthermore, technology has improved our ability to create conditions mimicking those existing inside biological membranes (the structures that separate the cell interior from its surroundings and organise the cell into compartments) greatly simplifying the process of obtaining crystals of proteins that are normally associated with membranes. Such proteins perform key biological functions at the cell surface, enabling cells to recognise one another and to bind biological surfaces, and regulating the traffic of molecules, including other proteins, into and out of the cell. However, membrane proteins are much harder to work with, and hence less well understood, than other protein systems.

Here we request funds to purchase equipment that will transform our ability to grow crystals, and obtain structures, of a range of biologically interesting but technically challenging targets. We will create a state-of-the-art Facility to exploit recent successes producing proteins and protein assemblies in the quantities necessary for crystallisation. Specifically, we wish to purchase: i) a robot to set up crystallisation experiments in conditions replicating the membrane environment; ii) an automated system to house the numbers of crystallisation experiments made possible by robotic systems working on small scales, and that will monitor their progress without human intervention; and iii) a complete crystallisation facility, including a robot to set up experiments and a microscope to inspect the results, maintained in a controlled, oxygen-free, environment. We will use this equipment to obtain structures of a number of biological molecules and assemblies including: the machinery controlling protein movement across membranes; the surface proteins of the human red blood cell that determine blood group, surface proteins from disease-causing bacteria that enable them to bind human cells; giant molecular machines synthesising drugs and antibiotics; the protein assembly by which cells carry out the instructions contained within genes; artificial proteins that carry electrons; and a wide range of proteins, involved in processes from bacterial antibiotic resistance to conversion of carbon dioxide into liquid fuels, that only function when oxygen is absent. Through our strong links to other local Universities our Facility, which will be unique within the region, will be open to researchers across the South West and South Wales, and will provide cutting edge instrumentation on which to provide the next generation of scientists with skills essential to the UK science and technology base.

Technical Summary

High-resolution structural information can transform our understanding of a given biological system. X-ray diffraction remains the technique with the broadest application to diverse biological targets. Advances in instrumentation at all stages of the macromolecular crystallography pipeline, from protein expression to software tools, have greatly expanded the range of targets that may be considered amenable to structure determination. In this application we seek support to equip a Facility with instrumentation to enable crystallisation of a range of challenging structural targets of high scientific value, including soluble and membrane protein complexes, de novo designed protein systems and oxygen-sensitive enzymes. We request funding for i) a lipidic cubic phase (LCP)-capable robot for crystallisation of membrane proteins and complexes; ii) an automated crystal incubation and imaging system able to maintain and monitor large numbers of experiments, with capability for fluorescence detection of protein crystals and microcrystals in conditions of high background (LCP) and iii) a dedicated anaerobic chamber housing a crystallisation robot and microscope, together with a port for crystal freezing, for crystallisation of oxygen-sensitive systems. Our targets include membrane protein complexes from pro- (protein export; bacterial surface protein) and eukaryotic (erythrocyte membrane proteins) sources, polyketide synthase and eukaryotic transcription super-complexes, de novo designed membrane channel and oxidoreductase systems and a range of radical and redox enzymes. The Facility will enable us to fully exploit recent progress generating material suitable for crystallisation and augment existing capabilities for recombinant protein production and characterisation. No similar equipment is installed in the South West, while our strong local links in structural biology and graduate student training will enable this to operate as a regional Facility with a wide user base.

Planned Impact

This proposed Centre will deliver a major new resource for structural biology, advancing our understanding of complex biological systems necessary for medical intervention and their modification and exploitation, which will contribute substantially to the UK's global leadership in these areas. The Centre will progress and diversify such studies, focusing on significant biological challenges that fit with the BBSRC World-Class Bioscience strategy. Moreover the Centre will impact upon important strategic areas of Synthetic Biology and Nanotechnology, by providing new high-resolution information of protein components and complex systems.

We intend that this research will impact upon policy-makers, funding bodies and academic institutions by providing clear evidence of the value of state-of-the-art equipment and fundamental, interdisciplinary research for the future of UK science and education. In order to deliver this impact we will highlight the importance of scientific knowledge in maintaining and increasing the UK's leading position in higher education, emphasising the central role of science in educating the public as well as in schools and lifelong learning. We will also continue to promote such research outside the University, in the South West, nationally and internationally, and discuss potential impact with colleagues, other institutions, funding bodies and learned societies. Moreover by increasing knowledge of basic science the Centre will have a major impact on interdisciplinary training of early-career researchers. Additionally it will facilitate outreach opportunities to educate the public and about the importance of scientific discovery and to inspire young students, revealing the intellectual benefits of a career in bioscience.

The Centre will also foster new projects and collaborations at Bristol and beyond, as well as providing a resource for researchers throughout the UK. It will also provide a hub for workshops and practical courses.

The main areas of impact will be:
a) Delivering a state-of-the-art facility for biological structure determination of complex biological systems, establishing a unique resource in the UK and enabling new collaborations and training opportunities.
b) Delivering scientifically literate people to society. School pupils, undergraduates, postgraduates and early career researchers.
c) Engaging in science festivals, schools visits, talks, debates and press briefings. School pupils have been very effectively involved to date, as have the public; we also plan to formulate new strategies to widen the impact.
d) In the longer term, realising impact in drug development, energy security and synthetic biology.
Description The award has led to the establishment of a University facility for the structural characterisation of oxygen sensitive proteins, which is now enabling fundamental insights into biocatalysis, antimicrobial resistance and de novo redox protein design. In addition, the imaging facilities are being used across the University to support research efforts in the study, design and exploitation of proteins, including a number of projects performed in collaboration with industry and in particular with collaborators in pharma. Multiple high-profile papers are soon to emerge thanks to this investment.

These facilities have proven instrumental in advancing structural analysis of de novo designed proteins (Woolfson & Brady labs). This has lead to substantial major advances including introducing enzymatic activity in a completely de novo heptad-barrel protein (Burton et al., Nature Chemistry 2016 DOI:10.1038/nchem.2555), rational design and assembly of a range of oligomeric and water-soluble alpha-helical barrels that form channels (Thompson et al, Science 2014 Vol 346:485-488 DOI: 10.1126/science.1257452) and self-assembling peptide-based nanotubes (Burgess et al, Journal of the American Chemical Society 2015 137 (33) pp 10554-10562 DOI: 10.1021/jacs.5b03973). These studies have all relied on detailed crystallographic structures which have only proven possible to obtain with the advanced crystallisation facilities provided from this grant.

The Anderson lab have deployed the anaerobic crystallisation facility in a study to identify and structurally characterise reactive intermediates in heme enzyme-catalysed carbene transfer reactions, where simple olefins are cyclopropanated using ethyldiazoacetate and ferrous hemoproteins. We have focussed initially on the crystallisation of several natural and engineered enzymes (horse heart myoglobin and an engineered variant of P450 BM3) in their reduced, air sensitive states. In an alternative approach, we have also crystallised in the air stable ferric forms and soaked with reductant in crystallo. The crystals of reduced hemoprotein are then soaked with ethyldiazoacetate and rapidly frozen in liquid nitrogen for subsequent X-ray data collection at Diamond Light Source. We hope this will provide a significant insight into the mechanism of this valuable abiological chemistry.

The high-throughput crystallization equipment was instrumental in the Berger lab to crystallize TBP associated factor (TAF) 2, a cornerstone of the super-complex that regulates the transcription initiation of all protein encoding genes in euakryotes. Initial hits resulted in crystals that already diffract synchrotron radiation to better than 4 Angstroem resolution. The facilities has help in getting optimized crystal diffracting to 2.4 Angstroem resolution. Structure determination is in progress.
Facilities have also been key in obtaining crystals of an oocyte specific transcription factor TBPL2, which diffracted to 2 Angstroem resolution. The structure was solved and coordinates have been deposited (PDB ID- 6TH9); the corresponding publication is about to be submitted.

Using these facilities, Berger lab have also obtained crystals of an engineered Crown Domain from ADDomer; a synthetic self-assembling platform for highly efficient vaccination. Crystals diffracted to 3 Angstroem resolution at diamond light source. Structure determination is in progress.

In the Collinson lab the centre has facilitated new projects for the structural analysis of a number of different proteins relevant to protein secretion, membrane protein insertion and the mechanism of bacterial antibiotic resistance. This work continues.

The Spencer group has solved a protein structures in anaerobic conditions using the crystallization set-up in the oxygen free glove box. They are not yet published; pdb ID 6FZ6.

The following structures (pubs and PDB code) that have been determined from collogues utilising the Bristol crystallisation facility and data collected at the Diamond beamlines:

Gupta, K., Berger, I. (2020)
Mouse TBP2 core domain (6TH9)

Race, P. (2020) 6SZC currently unreleased

Kamsri et al (2020) JCIM PubMed 31820972 PDB 6R9W

Tooke et al RSC Med Chem (2020) doi 10.1039/C9MD00557A PDBs 6TD0, 6TD1

Bicyclic Boronate VNRX-5133 Inhibits Metallo- and Serine-ß-Lactamases.
Krajnc A, Brem J, Hinchliffe P, Calvopina K, Panduwawala T, Lang PA, Kamps JJAG, Tyrell JM, Widlake E, Saward BG, Walsh TR, Spencer J, Schofield CJ.
J Med Chem. 2019 Aug 27. doi: 10.1021/acs.jmedchem.9b00911. PDB code: 6RMF

Molecular Basis of Class A ß-lactamase Inhibition by Relebactam.
Tooke CL, Hinchliffe P, Lang PA, Mulholland AJ, Brem J, Schofield CJ, Spencer J.
Antimicrob Agents Chemother. 2019 Aug 5. pii: AAC.00564-19. doi: 10.1128/AAC.00564-19.
PDB codes: 6QWE, 6QWD, 6QWC, 6QWB, 6QWA, 6QW9, 6QW8, 6QW7

In prep: Lythell et al "Formation of the reaction intermediate in MCR bacterial phosphoethanolamine transferases responsible for transmissible colistin antibiotic resistance". PDB code: 6SUT

Structure and mechanism of a dehydratase/decarboxylase enzyme couple involved in polyketide ß-methyl branch incorporation. Nair AV, Robson AR, Akrill T, Till M, Byrne MJ, Tiwari K, Willis CL and Race PR. JBC in revision. PDBs. 4Q1K, 4Q1G, 4Q1J, 4Q1HA

A Natural Diels-Alder Biocatalyst Enables Efficient [4 + 2] Cycloaddition Under Harsh Reaction Conditions. Marsh CO, Lees NR, Han L-C, Byrne MJ, Mbatha SZ, Maschio L, Pagden-Ratcliffe S, Duke PW, Stach JEM, Curnow P, Willis CL, and Race PR. ChemCatChem, in press, DOI 10.1002/cctc.201901285

Bicyclic Boronate VNRX-5133 Inhibits Metallo- and Serine-ß-Lactamases. Krajnc A, Brem J, Hinchliffe P, Calvopiña K, Panduwawala TD, Lang PA, Kamps JJAG, Tyrrell JM, Widlake E, Saward BG, Walsh TR, Spencer J, Schofield CJ J Med Chem. 2019, in press doi: 10.1021/acs.jmedchem.9b00911.

Molecular Basis of Class A ß-lactamase Inhibition by Relebactam. Tooke CL, Hinchliffe P, Lang PA, Mulholland AJ, Brem J, Schofield CJ, Spencer J. Antimicrob Agents Chemother. 2019. pii: AAC.00564-19. doi: 10.1128/AAC.00564-19.

Thomas, F.; Dawson, W.; Lang E.J.M.; Burton, A.; Bartlett, G.J.; Rhys, G.G.; Mullholand, A.J.; Woolfson, D.N. (2018). 
De Novo-Designed a-Helical Barrels as Receptors for Small Molecules ACS
Synthetic Biology 7, 1808, doi: 10.1021/acssynbio.8b00225 
[PDB ID: 6EIK and 6EIZ]

Rhys, G.G.; Wood, C.W.; Lang E.J.M.; Mulholland, A.J.; Brady, R.L.; Thomson, A.R.; Woolfson, D.N. (2018).
Maintaining and breaking symmetry in homomeric coiled-coil assemblies 
Nature Commun In Press, (2018). DOI : 10.1038/s41467-018-06391-y
[PDB ID: 6G65, 6G66, 6G67, 6G68, 6G69, 6G6A, 6G6B, 6G6C, 6G6D, 6G6E, 6G6F, 6G6G and 6G6H]

Wang L, Parnell A, Williams C, Bakar, N. A., Challand, M. R., van der Kamp M. W., Simpson,T. J., Race P. R., Crump,M. P., Willis, C. L.
A Rieske oxygenase/epoxide hydrolase-catalysed reaction cascade creates oxygen heterocycles in mupirocin biosynthesis. 
Nature Catalysis, (2018), accepted.

Tetrahydropyran formation in the biosynthesis of the antibiotic mupirocin proceeds via an oxygenase/epoxide hydrolase cascade.
Luoyi Wang, Alice Parnell, Christopher Williams, Nurfarhanim A. Bakar, Marc W. van der Kamp, Thomas J. Simpson, Paul R. Race, Matthew P. Crump, Christine L. Willis.
In review - Nature Chemistry [PDB ID: 6FXD]

Structural and Kinetic Studies of the Potent Inhibition of Metallo-ß-lactamases by 6-Phosphonomethylpyridine-2-carboxylates. Philip Hinchliffe, Carol A. Tanner, Anthony P. Krismanich, Geneviève Labbé, Valerie J. Goodfellow, Laura Marrone, Ahmed Y. Desoky, Karina Calvopiña, Emily E. Whittle, Fanxing Zeng, Matthew B. Avison, Niels C. Bols, Stefan Siemann, James Spencer, and Gary I. Dmitrienko.
Biochemistry, 2018, 57 (12), pp 1880-1892. DOI: 10.1021/acs.biochem.7b01299. [PDB ID: 5HH5, 5HH6, 5HH4]
Cyclobutanone Mimics of Intermediates in MetalloßLactamase Catalysis.
Dr. Martine I. Abboud Dr. Magda Kosmopoulou Dr. Anthony P. Krismanich Dr. Jarrod W. Johnson Dr. Philip Hinchliffe Dr. Jürgen Brem Prof. Dr. Timothy D. W. Claridge Dr. James Spencer Prof. Dr. Christopher J. Schofield Prof. Dr. Gary I. Dmitrienko.
Chemistry. 2018. DOI: 10.1002/chem.201705886 [Epub ahead of print]. [PDB: 5NDB and 5NDE]
Crystal Structure of a radical SAM methyltransferase from Sphaerobacter thermophiles.
Shaw, J., Hinchliffe, P., Paterson, N., Challand, M., Spencer, J.
In prep [PDB ID: 6FZ6]

De novo designed a-helical barrels as receptors for small molecules
Thomas, F.; Dawson, W.; Lang E.J.M.; Burton, A.; Bartlett, G.J.; Rhys, G.G.; Mullholand, A.J.; Woolfson, D.N. (2017)
In prep [PDB ID: 6EIK and 6EIZ]

Baxter D, Perry SR, Hill TA, Kok WM, Zaccai NR, Brady RL, Fairlie DP, Mason JM. (2017) Downsizing Proto-oncogene cFos to Short Helix-Constrained Peptides That Bind Jun. ACS Chem Biol. 2017 Aug 18;12(8):2051-2061. doi: 10.1021/acschembio.7b00303 [PDB ID: 5FV8]

Nicholas R. Lees, Li-Chen Han, Matthew J. Byrne, Jonathan A. Davies, Alice E. Parnell, Pollyanna E. J. Moreland, James E. M. Stach, Marc W. van der Kamp, Christine L. Willis, and Paul R. Race (2017). A Bifunctional Lyase-Esterase Catalyzes Acetate Elimination in Spirotetronate/Spirotetramate Biosynthesis. In prep [PDB ID: 5NO5 and 4YWF]

Thomas, F.; Dawson, W.; Lang E.J.M.; Burton, A.; Bartlett, G.J.; Rhys, G.G.; Mullholand, A.J.; Woolfson, D.N. (2017) In prep [PDB ID: 6EIK and 6EIZ]

Calvopiña K, Hinchliffe P, Brem J, Heesom KJ, Johnson S, Cain R, Lohans CT, Fishwick CWG, Schofield CJ, Spencer J, Avison MB. (2017) Structural/mechanistic insights into the efficacy of nonclassical ß-lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates. Mol Microbiol. 2017 Sep 6. doi: 10.1111/mmi.13831. [Epub ahead of print] PMID: 28876489 [PDB ID: 5NE1 5NE2 5NE3]

Coates K, Walsh TR, Spencer J, Hinchliffe P. (2017) 1.12 Å resolution crystal structure of the catalytic domain of the plasmid-mediated colistin resistance determinant MCR-2.
Acta Crystallogr F Struct Biol Commun. 2017 Aug 1;73(Pt 8):443-449. doi: 10.1107/S2053230X17009669. Epub 2017 Jul 26.
PMID: 28777086 [PDB ID: 5MX9]

Reichenbach, Linus F., Sobri, Ahmad Ahmad, Zaccai, Nathan R., Agnew, Christopher, Burton, Nicholas, Eperon, Lucy P., de Ornellas, Sara, Eperon, Ian C., Brady, R. Leo., Burley, Glenn A. Structural Basis of the Mispairing of an Artificially Expanded Genetic Information System. Cell Chem. (1) 946-958 (2016) [PDB id 5LJ4, 5I4S, 5KTV]

Burton, A. J., Thomson, A. R., Dawson, W. M., Brady, R. L. & Woolfson, D. N. Installing hydrolytic activity into a completely de novo protein framework. Nat. Chem. 8, 837-844 (2016). PDB codes: 5EZA, 5EZC, 5EZE, 5EZ8, 5EZ9 & 5F2Y.

The Streptococcus gordonii Adhesin CshA Protein Binds 2. 2, Host Fibronectin via a Catch-Clamp Mechanism. Back CR, Sztukowska MN, Till M, Lamont RJ, Jenkinson HF, Nobbs AH, Race PR. J Biol Chem. (2017) Feb 3;292(5):1538-1549. doi: 10.1074/jbc.M116.760975. 5L2D

Structural and Functional Analysis of Cell Wall-anchored Polypeptide Adhesin BspA in Streptococcus agalactiae. Rego S, Heal TJ, Pidwill GR, Till M, Robson A, Lamont RJ, Sessions RB, Jenkinson HF, Race PR, Nobbs AH. J Biol Chem.(2016) Jul 29;291(31):15985-6000. doi: 10.1074/jbc.M116.726562. 4Z23, 4Z1P

The Catalytic Mechanism of a Natural Diels-Alderase Revealed in Molecular Detail. Byrne MJ, Lees NR, Han LC, van der Kamp MW, Mulholland AJ, Stach JE, Willis CL, Race PR. J Am Chem Soc. (2016) May 18;138(19):6095-8. doi: 10.1021/jacs.6b00232. 5DYV, 5DYQ

Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist. Frago S, Nicholls RD, Strickland M, Hughes J, Williams C, Garner L, Surakhy M, Maclean R, Rezgui D, Prince SN, Zaccheo OJ, Ebner D, Sanegre S, Yu S, Buffa F, Crump MP and Hassan AB Proc. Natl. Acad. Sci. (2016) 113, 2766-2775. 5IEI

A Bifunctional Lyase-Esterase catayzes Acetate Elimination in Spirotetronate/Spirotetramate biosynthesis Lees N, Han L-C, Byrne MJ, Davies, JA, Parnell AE, Moreland PE, Stach JEM, van der Kamp M, Willis CL and Race PR. (2017) Submitted Angew Chem Int Ed Engl. 4YWF

Crystal structures of MmgE/PrpD polypeptides from Bacillus subtilis and Salmonella enterica reveal the molecular basis of 2-methylcitrate dehydration in a ubiquitous enzyme superfamily. Baker GE, Marsh CO, van der Kamp MW and Race PR. (2017) Submitted J. Biol. Chem. 5MVI, 5MUX

Redox control of bacterial sporulation revealed by the structure of the 3-ketoacyl CoA thiolase MmgA. Baker GE and Race PR. (2017) Submitted J. Mol. Biol. 5LP7

Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1. Hinchliffe P, Yang QE, Portal E, Young T, Li H, Tooke CL, Carvalho MJ, Paterson NG, Brem J, Niumsup PR, Tansawai U, Lei L, Li M, Shen Z, Wang Y, Schofield CJ, Mulholland AJ, Shen J, Fey N, Walsh TR, Spencer J. Sci Rep. 2017 Jan 6;7:39392. doi: 10.1038/srep39392. PDB ids 5LRM, 5LRN
This paper/these structures also highlighted on Diamond website: http://www.diamond.ac.uk/Science/Research/Highlights/2017/MX-resistance.html

F-NMR Reveals the Role of Mobile Loops in Product and Inhibitor Binding by the São Paulo Metallo-ß-Lactamase. Abboud MI, Hinchliffe P, Brem J, Macsics R, Pfeffer I, Makena A, Umland KD, Rydzik AM, Li GB, Spencer J, Claridge TD, Schofield CJ. Angew Chem Int Ed Engl. 2017 Mar 27;56(14):3862-3866. doi: 10.1002/anie.201612185. PDB id 5LS3

Cross-class metallo-ß-lactamase inhibition by bisthiazolidines reveals multiple binding modes. Hinchliffe P, González MM, Mojica MF, González JM, Castillo V, Saiz C, Kosmopoulou M, Tooke CL, Llarrull LI, Mahler G, Bonomo RA, Vila AJ, Spencer J.
Proc Natl Acad Sci U S A. 2016 Jun 28;113(26):E3745-54. doi: 10.1073/pnas.1601368113.
PDB ids 5EW0 5EWA 5EV6 5EV8 5EVB 5EVD 5EVK

Structural and Biochemical Characterization of Rm3, a Subclass B3 Metallo-ß-Lactamase Identified from a Functional Metagenomic Study. Salimraj R, Zhang L, Hinchliffe P, Wellington EM, Brem J, Schofield CJ, Gaze WH, Spencer J. Antimicrob Agents Chemother. 2016 Sep 23;60(10):5828-40. doi: 10.1128/AAC.00750-16. Print 2016 Oct. PDB id 5IQK
Exploitation Route This facility is now in integral part of BrisSynBio equipment suite, which will help sustain and update the centre for structural analysis in Bristol.

The fully functional and future proofed facility is now available to all structural biologists at Bristol for FREE. So, we now anticipate that it will this access will bolster structural biology at Bristol and increase our capabilities for protein structure determination. This is more relevant now than ever. We have secured the funds from the Wellcome Trust and from GW4 partners the £2.5M to establish high resolution electron microscopy and BBSRC funded computer cluster for image processing (BB/R000484/1 BlueCryo Image Processing Computing Cluster). The combination of electron microscopy of large molecular assemblies with X-ray crystallography of individual components and sub-assemblies will prove very powerful indeed.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Wellcome Trust Multi-user equipment grant - South West Regional Facility for High-Resolution Electron Cryo-Microscopy
Amount £1,000,000 (GBP)
Funding ID 202904 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2016 
End 05/2021