Enrollement and places of work: CSSB, DESY, LIV, University of Groningen, University of Siegen

PhD Project 1

Sample delivery for SPI: protein orientation and detection

Enrolment in Doctoral degree: University of Siegen, Siegen, Germany

Place of work: Centre for Structural Systems Biology, Hamburg, Germany

Supervisor: Dr. Thomas Kierspel and Prof. Dr. Charlotte Uetrecht

X-ray free electron lasers (XFELs) are the brightest X-ray sources in the world and are able to shine new light in the complex dynamics of biological systems. They enable imaging techniques like Single-particle imaging (SPI), which allows to retrieve high structural information (atomic resolution) from protein samples without the need for crystallisation, i.e., a key limitation of current X-ray imaging techniques. Despite its potential, SPI is still in its early stages, and development is needed to make it a useful tool for structural biology. Within this project tools will be developed to improve the sample delivery system for SPI, with a focus on the mass-spectrometry based sample delivery system itself, and the orientation of proteins in external electric fields (DC, laser). Experiments will be conducted in laboratory setup in Hamburg, as well as at X-ray sources like the European XFEL, FLASH, or PETRA III synchrotron. Further scientific and engineering boundaries for experiments at the TXI instrument (LCLS II, USA) will be elaborated. This project encompasses design, set up and execution of novel scientific experiments. Intense collaborative work between different partners is very important, and secondments are planned at MS Vision (NL), LCLS II (USA), and University Uppsala (SE) covering basic MS techniques, X-ray matter interaction, and data analysis for X-ray diffraction experiments like SPI.

PhD Project 2

MSN and SPI of coronaviral nsp complexes for structural investigations

Enrolment in Doctoral degree: University of Siegen, Siegen, Germany

Place of work: Centre for Structural Systems Biology, Hamburg, Germany

Supervisor: Prof. Dr. Charlotte Uetrecht

Viral protein complexes are highly dynamic entities shuttling between different states with distinct function. This holds also true for the replication/transcription complexes (RTCs) of coronaviruses, which are built up form 9 individual non-structural proteins (nsps). Understanding the RTCs is crucial for drug development and unravelling functional aspects of the viral lifecycle. In this project, conformational states and their dynamic interchanges in RTCs from multi-species mixtures of controlled composition will be investigated. An Orbitrap UHMR with Omnitrap will be used to do multi-stage fragmentation with UV photodissociation and high energy collision dissociation. Thereby, exposed residues can be identified in native top-down MS. These experiments will be complemented with X-ray induced fragmentation, single particle imaging (SPI) and SAXS in the gas phase of mass- and conformer-selected species on a modified prototype setup developed in MS SPIDOC. This project will encompass acquisition, analysis and curation of native top-down MS data; combination of structural MS data with gas phase structures and modelling to understand conformational dynamics as well as protein production. This project involves regular exchange with Uppsala University and IMIC in Prague. Furthermore, secondments are foreseen within the SPIDocs consortium to obtain advanced Omnitrap training at Fasmatech (Gr), additional insights to protein production and characterization at Evotec (UK) and to develop data standards for native top-down MS with EBI (UK).

Image from Krichel B. et al., Biochem J, 2020

PhD Project 4

Gas-phase photodissociation processes of relevance for imaging biomolecules and understanding protein drug interaction

Enrolment in Doctoral degree: University of Groningen, Groningen, Netherlands

Place of work: Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany

Supervisor: Prof. Dr. Sadia Bari

To enhance the understanding of protein drug interactions action spectroscopy at specific heteroatoms in peptides and proteins will be used to study interactions with metals and salts. The doctoral candidate will pursue recent observations about electronic structures and molecular transitions in particular by applying near-edge X-ray absorption mass spectrometry (NEXAMS), an action spectroscopy technique based on resonant photoexcitation and photoionisation of atomic core levels and measurement of ionised species and dissociation products as a function of X-ray photon energy.  Another aspect of the project is to extend current research in single particle imaging (SPI) and action spectroscopy by combining both techniques using fragmentation and spectroscopy techniques at different wavelength ranges in experiment and theory. The doctoral candidate would learn how to prepare and perform experiments at light source facilities, also in larger and international teams, how to perform action spectroscopy in combination with MS, and how to analyse and interpret the acquired data with theoretical data. There will be a mutual exchange between theory and experiment to understand the electronic structure and fragmentation patterns in biomolecules and how this can be used for SPI, for which a secondment to Uppsala University is planned. Complementary work on characterization of photo-fragmentation paths at low photon energies and structural analysis is done together within the SPIDocs consortium by further secondments at University Claude Bernard Lyon and VU Amsterdam.

Image from J. Am. Soc. Mass Spectrom. 2021 32 (3), 670-684.

For questions related to application to University of Siegen

For questions related to application to University of Groningen