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Contenuto archiviato il 2024-06-18

Membrane Protein Nanocrystallography

Final Report Summary - NANOMEM (Membrane Protein Nanocrystallography)

A SUMMARY DESCRIPTION OF THE PROJECT OBJECTIVES

NanoMem (“Membrane Protein Nanocrystallography”) has been a very successful collaborative project and we are very grateful for this investment in a rapidly developing area of life-science.

Modern structural biology builds upon synergies between lab-bench scale science on the one hand and large scale research infrastructure on the other. NanoMem recognised the transformative opportunities created by current X-ray source and detector developments to impact strongly on membrane protein structure, a challenging sub-field of structural biology. We have applied synchrotron based micro-focus X-ray beams to address challenging diffraction studies from small membrane protein crystals and have taking an international lead in realizing new possibilities created by X-ray Free Electron Lasers to deliver an entirely new regime of high-resolution serial femtosecond crystallography of membrane proteins. Our interdisciplinary and intersectorial research training work programme incorporated membrane protein production, purification and crystallisation, micro and nano-crystal manipulation, micro-focus diffraction at synchrotron sources, nano-focus diffraction at X-ray free electron lasers, serial femtosecond crystallography of static and time-resolved systems, software development, inhibitor binding studies, and commercialisation of the most helpful innovations.

NanoMem consisted of eleven Partner Institutes, of which ten in the original contract and the University of Oxford was included in an amendment to the contract. Two are in Sweden (University of Gothenburg, UGOT; AstraZeneca, AZ), three are in the United Kingdom (Imperial College London, IMPERIAL; Molecular Dimensions Lts, MDL; University of Oxford, UOXF), two are in Switzerland (Paul Scherrer Institute, PSI; SwissSci AG; SWISSCI), two are in Germany (University of Hamburg, UHAM; Max Planck Institute Heidelberg, MPI) and two are in France (European Synchrotron Radiation Facility, ESRF; Centre National de la Recherche Scientifique, CNRS).

A DESCRIPTION OF THE WORK PERFORMED SINCE THE BEGINNING OF THE PROJECT

We have had the pleasure of educating a talented and motivated group of young scientists in a diverse base of skills. In total thirteen ESRs and one ER have been trained within NanoMem. The sole ER (Jonna Hakulinen, AZ) completed her full quota of 24 months training. Four ESRs (Rob Dods, UGOT; Wenting Wu, PSI; Aleksandra Wanda Woznicka; CNRS; and Marcus Metz, UHAM) completed their full allocation of 36 months of training within NanoMem and Anastasya Shilova (ESRF) completed 35.75 months of training due to starting her position a few days into January 2014. Ziva Vuckovic and Katarina Jungnickel changed from training within industrial partners (SWISSCI and MDL respectively) to academic insititutions (PSI and UOXF respectively) during their training within NanoMem and thus received 34 and 30 months training in total within the contract. Ana Rita Santos (IMPERIAL, 26.55 months training), Frank Lehmann (IMPERIAL, 6.8 months training) and Mohd Akram (MPI 12 months training) decided to leave the project early. Gabriela Kovacsova (MPI, 24 months training), Florian Schmidt (UGOT, 3.9 months training) and Tamara Muji (PSI, 5 months training) were offered shorter appointments to complete the objectives of the contract.

The NanoMem mid-term meeting took place on November 6th at Sheraton Airport Hotel, Brussels. The project officer signalled how the project should conform to the Marie Curie Framework programme and what steps needed to be taken by SWISSCI, MDL, UOXF and PSI to conform to requirements that each ESR was working full time at the place of employment. This led to a change in the contract with UOXF becoming a full partner.

A DESCRIPTION OF THE MAIN RESULTS ACHIEVED SO FAR

All ERs and ESRs were engaged in the research objectives of this project and there was considerable interaction between partners and a lively participation in the training programme. We have trained these young scientists across disciplines of membrane protein production, crystallization and X-ray diffraction. Our collaboration with industry has produced new membrane protein structures in complex with small molecule compounds, exploration of materials for crystallization, and a range of crystallisation products. We have delivered a complete training programme with events throughout the grant period and with good participation in the NanoMem events by the young scientists appointed to the contract. We have helped develop the field of microcrystallography using serial crystallography techniques. This has been an exciting time of rapid change to the field and the ER and ESRs have developed a portfolio of skills that are valuable for their future careers. We have produced a large number of publications, several of which were published in Nature, Science and Nature daughter journals with a contribution from those trained within the project. Some scientific highlights to which ERs or ESRs employed on NanoMem contributed to the work include:

Nogly P et al., Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography. Nature Commun. 7, 12314 (2016).
Nango, E., et al. A three-dimensional movie of structural changes in bacteriorhodopsin. Science 354, 1552-1557 (2016).
Barends, T.R.M et al., Direct Observation of Ultrafast Collective Motions in Carbonmonoxy Myoglobin Upon Ligand Dissociation, Science 350, 445-450 (2015).
Ayyer, K. et al., Macromolecular diffractive imaging using imperfect crystals. Nature. 530, 202-6 (2016).
Pande, K. et al., Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein. Science. 352, 725-9 (2016).
Hakulinen, J. et al. MraY-antibiotic complex reveals details of tunicamycin mode of action.
Nature Chem. Biol. 13, 265–267 (2017)

THE EXPECTED FINAL RESULTS AND THEIR POTENTIAL IMPACT AND USE (INCLUDING THE SOCIO-ECONOMIC IMPACT AND THE WIDER SOCIETAL IMPLICATIONS OF THE PROJECT SO FAR)

Serial Crystallography methods developed within the framework of this grant has become mainstream and is widely used by many scientists at X-ray free electron laser facilities and increasingly at synchrotron radiation facilities. UHAM have made software publically available for scientists to use and this has greatly facilitated the widespread growth of this method. This will be a very long-term and international impact of this project. MDL lead our work towards the commercialization of our research. ESR 7 (Katharina Jungnickel) tested a variety of products for Molecular Dimensions Ltd in close collaboration with UOXF and a number of items have been released for sale.