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Mass Spectrometry for Single Particle Imaging of Dipole Oriented protein Complexes

Periodic Reporting for period 2 - MS SPIDOC (Mass Spectrometry for Single Particle Imaging of Dipole Oriented protein Complexes)

Reporting period: 2019-09-01 to 2022-05-31

The European XFEL with its unparalleled peak brilliance and repetition rate has the potential to further applications in single particle imaging (SPI), thus far limited to large viral particles at X-ray Free-Electron Lasers (XFEL). SPI will allow imaging protein complexes without the need for crystallization. This eventually renders transient conformational states accessible for high resolution structural studies yielding molecular movies of biomolecular machines. A major bottleneck is the wealth of data required to reconstruct a single structure leading to long processing times.
MS SPIDOC will overcome this data challenge by developing a native mass spectrometry (MS) system for sample delivery, named X-MS-I. It will provide mass and conformation selected biomolecules, which are oriented along their dipole axis upon imaging. This will enable structural reconstructions from much smaller datasets speeding up the analysis time tremendously. Moreover, the system features low sample consumption and a controlled low background easing pattern identification.
The main objectives of the project are:
• Deliver mass and conformation separated biomolecules for SPI.
• Orient proteins for SPI.
• Image protein complex unfolding.
• Exploit potential of protein orientation for other applications.
The MS SPIDOC consortium combines internationally leading expertise in different fields relevant to the project. First designs of components for mass and conformation selection as well as efficient sample introduction are currently being tested. Results are in line with our requirements. Protein orientation is investigated theoretically. Electric fields theoretically facilitating dipole orientation have been achieved on an orientation device. We expect that the device can benefit other gas phase applications and are investigating the potential. A technical design report for implementation of the prototype at European XFEL has been filed. Individual components will be assembled in the next period and tested prior to SPI experiments.
MS SPIDOC aims at developing a new type of sample environment for structural biology to be installed at the SPB/SFX instrument of the European XFEL. MS SPIDOC is structured into four WPs, including a management/ exploitation/ dissemination WP responsible for smooth interaction and communication between the partners and three research-focused WPs. WP1 focusses on establishing dipole orientation of protein ions. This includes simulation of charge distributions in ESI, timeframe for orientation in a field for large proteins under varying conditions (gas, field strength etc.). The results will directly feed into device development and subsequent testing. In WP2, additional components are developed and then combined into the X-MS-I prototype. Activities mainly focus on three parts of the instrument ensuring: i) ideal ion production and collection at the source (aerolens); ii) mass selection and trapping (digital ion trap) and iii) the ability to sort ions by shape (high fidelity IM). This ensures that enough proteins are available for the interaction with the beam and streamlined data analysis. Ion optical trajectories are simulated to facilitate smooth development. The results will lead to integration of the prototype into the SPB/SFX instrument including the control system. Furthermore, the experiment will be simulated for benchmarking and comparison to recorded data. WP2 is tightly connected to WP3. While WP2 comprises the practical building of the devices and the prototype, WP3 is responsible for benchmarking and testing of components to ensure structural integrity of protein complexes. Furthermore, the entire prototype will be tested culminating in first SPI experiments. Data analysis will also be part of WP3. Results of benchmarking will feed into further optimization of components and the prototype.
The first 4 scientific deliverables have been submitted:
D1.1 Stability range and charge distribution of electrosprayed ions
D2.1 Ion optical models of devices
D2.2 Construction of transfer interface & digital trap
D3.1 Instrumental settings for structural integrity
D1.1 and D3.1 also feed into milestone 1, evaluation of structure conservation. Based on available IM and action-FRET data as well as simulations, we expect that protein structures are sufficiently stable in the gas phase. The results will be extended to a range of standards in order to select the best settings for unknown samples based on similar sized standards.
First generations of components (aerolens, digital filter and ion trap, ion mobility, orientation device) have been assembled and are undergoing testing now. In sum, component development and testing for X-MS-I is well under way. Moreover, recommendations for structural preservation have been defined and will be extended in the near future. Protein orientation indeed appears feasible and we plan on exploiting its potential for other applications soon.
So far, the project is well on track. The developed and tested components show good functionality with respect to required specifications. Hence, we expect to indeed assemble a working prototype and use it for SPI experiments at EuXFEL before the end of the project. Based on theory and high voltage tests, dipole orientation has high chances for success. Moreover, UU and HPI intend to exploit dipole orientation for gas phase SAXS in the near future. The overall expected impact as well as an update on identified risks is presented below.
First publications are in preparation. Main impact is expected in the second period, when X-MS-I prototype becomes available and designed components will be exploited for marketing potential.