Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - X-probe (Advanced XFEL and Synchrotron based Probes of Protein Structure and Dynamics)

Teaser

In the cross-disciplinary X-Probe project we have built upon the opportunities created by current X-ray sources and of the new ones recently made operative in Europe and worldwide. We have timely trained a new generation of scientists for the present generation of advanced...

Summary

In the cross-disciplinary X-Probe project we have built upon the opportunities created by current X-ray sources and of the new ones recently made operative in Europe and worldwide. We have timely trained a new generation of scientists for the present generation of advanced X-ray facilities. Scientifically, X-probe has impacted on our understanding of protein conformational dynamics from femtoseconds to seconds, one of the most challenging problems in structural biology. We have unveiled the structural dynamics of protein that constitute relevant targets in pharmacology and nanotechnology. X-Probe has advanced the technological field of serial approaches at synchrotron radiation facilities by developing software and hardware solutions that extend the revolutionary possibilities created by X-ray Free Electron Lasers, which combine ultrafast X-ray pulses with high brilliance focusing capabilities.
Exploitation of these changing technical capabilities required interdisciplinary collaboration between structural biologists, physical chemists, beamline engineers, software developers and industrial partners. Rapid changes in the state-of-the-art demanded young researchers to be trained to meet new experimental, technical and analysis challenges at the forefronts of structural biology. X-probe addressed this challenge through an interdisciplinary and intersectorial training network incorporating protein micro-crystallisation, sample manipulation, beamline development, micro-focus diffraction, software development, time-resolved diffraction, time-resolved wide angle X-ray scattering, and serial femtosecond crystallography. On-site scientific training and seamless industrial participation was complemented with a scientific mentoring program and training in areas such as strategic project planning, intellectual property, commercialisation and communication skills. We delivered a young team of motivated researchers trained to develop new tools for extracting new insights in protein structural dynamics from femtoseconds to second that exploit the extreme X-ray brilliance of recently developing X-ray user facilities.
The very relevant European investments into the European XFEL and SwissFEL as well as the ongoing upgrade of synchrotron radiation sources demanded training of young scientists to fully exploit the scientific and technological avenues opened by this European and national effort, we developed a synergistic approach with technologically advanced enterprises to allow ESRs to convert knowledge and ideas into products and services for society and their own benefit.

Work performed

The Network has striven towards the study of protein dynamics, the development of state-of-the-art infrastructures and software suites for the use of the European and global scientific and industrial community and for the development of the Industrial Partners.
The main results are: advancements in the production of integrated software suites for time resolved protein studies at synchrotron and XFEL, production of engineered proteins for theranostics and their studies by time resolved methods at synchrotron and XFELs, determination of the structure of crucial drug targets (antibiotics and GPCRs), commissioning of time resolved synchrotron sources and implementation of new methods for data collection and analysis at existing ones, development of methodologies for production and analysis of micro and nano-crystals.
Ten ESRs have been recruited enhancing transnational mobility in the EU and attracting brilliant young scientists with outstanding potential from non-EU Countries. Nine training events took place on ”Frontier methods for visualizing proteins structural dynamics and their applications”, “Protein Structural dynamics: biological systems and experimental strategies”, “Commercialization of science”, “Project management and transferable skills” and “Serial crystallography at FELs and synchrotrons”, “The role of science in society, public outreach, career development and gender issues, “ Drug discovery from a structure and biophysics perspective” and a final Conference on “Novel Methods for Biomolecular Structure Determination”. Some of the training events included hands-on training. The ESRs have formed a team and are in contact, with a positive and supportive attitude, they have benefited of several joint experimental sessions. Collaborations have been cemented and new collaborations have been established also at the intersectorial level.
Dissemination activities have been carried out, at about 30 scientific events, including presentations by the ESRs. Outreach activities to communicate about the network, the scientific relevance of protein dynamics studies at synchrotron and XFEL and about MSCA-H2020 actions included an events targeted to schools in France, Italy, Germany and Switzerland, events at Open Door Days at synchrotrons (Hamburg, Villigen, ESRF) and a talk given by one of the ESRs at a Marie Curie Day (Gothenburg).

Final results

The action has led to a diverse research network, with synergy for developing research and training of young researchers to exploit the new frontiers of protein dynamics studies opened by the new generation X-ray sources in Europe and worldwide.
More in detail, we have obtained advancement in the study of the dynamics of protein in solution by carrying out studies at the XFEL of neuroglobin, myoglobin and hemoglobin. We have also studied the assembly of engineered and wild type archaeal ferritins with time resolved small and wide-angle X-ray scattering.
The study of more challenging membrane proteins has progressed with the determination of the structure of jumping spider rhodopsin, a GPCR that has unique properties that will allow us to study, for the first time, the time resolved structural dynamics of opsins.
Crystals of sensory rhodopsin II (SRII) and the transducer subunit HtrII have been obtained, which will widen our understanding the time evolution of bacteriorhodopsin thanks to the different time scales spanned by this system in its activity cycle. The determination of the structure of the antibiotic target phospho-N-acetylmuramoyl–pentapeptide translocase (MraY) in complex with the nucleoside antibiotic tunicamycin constituted a crucial breaktrough, with implications on the design of a new generation of antibiotics, a strategic task to address drug-resistance. The project has yielded advancements in developing software for XFEL and serial crystallography data analysis and for the treatment of time resolved WAXS data. Last but not least, the commissioning of the time resolved beam line at MAXIV was concluded and the time-resolved beam line ID09 at ESRF has been extensively used for training and for experimental sessions of the X-Probe scientific projects, implementing a rapid-mixing apparatus, eliminating the requirement of light-triggered reactions.
To summarize, the X-Probe ITN has contributed to the understanding of the evolution of protein structure in time utilizing the most advanced X-ray sources available. We have studied model systems such as globins and rhododpsins and we are also studying systems that will lead to progress in the development of new drug carriers (ferritins), in drug design (MraY) and in understanding the transmission of the light signal in the eye (sensory rhodopsin). We have also developed methods, software and contributed to the development of advanced infrastructures in the field in European facilities.

Website & more info

More info: http://www.x-probe.org.