MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm-1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

Centre for Free Electron Laser Science Deutsches Elektronen Synchrotron DESY Notkestraße 85 E22607 Hamburg Germany

Centre for Structural Systems Biology Deutsches Elektronen Synchrotron DESY Notkestraße 85 22607 Hamburg Germany

Department of Chemistry BMC Uppsala University Box 576 75123 Uppsala Sweden

Department of Physics and Astronomy Uppsala University Box 516 75120 Uppsala Sweden

European XFEL Holzkoppel 4 22869 Schenefeld Germany

Faculty 5 School of Life Sciences University of Siegen Adolf Reichwein Str 2a 57076 Siegen Germany

Fasmatech Technological and Scientific Park of Attica Lefkippos NCSR DEMOKRITOS Patr Gregoriou E' 27 Neapoleos Str 153 41 Agia Paraskevi Attica Greece

Institut Für Physik Universität Greifswald Felix Hausdorff Str 6 17489 Greifswald Germany

Institute of Microbiology of the Czech Academy of Sciences BIOCEV Průmyslová 595 Vestec 252 50 Czech Republic

Leibniz Institute of Virology Martinistraße 52 20251 Hamburg Germany

Manchester Institute of Biotechnology and Department of Chemistry The University of Manchester Manchester M1 7DN UK

MS Vision Televisieweg 40 1322 AM Almere Netherlands

University of Applied Sciences Technikum Wien Höchstädtpl 6 1200 Vienna Austria

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X-ray spectroscopy meets native mass spectrometry: probing gas-phase protein complexes