Segmented flow generator for serial crystallography at the European X-ray free electron laser

Echelmeier, A; Villarreal, J Cruz; Messerschmidt, M; Kim, D; Coe, JD; Thifault, D; Botha, S; Egatz-Gomez, A; Gandhi, S; Brehm, G; Conrad, CE; Hansen, DT; Madsen, C; Bajt, S; Meza-Aguilar, JD; Oberthür, D; Wiedorn, MO; Fleckenstein, H; Mendez, D; Knoška, J; Martin-Garcia, JM; Hu, H; Lisova, S; Allahgholi, A; Gevorkov, Y; Ayyer, K; Aplin, S; Ginn, HM; Graafsma, H; Morgan, Andrew J; Greiffenberg, D; Klujev, A; Laurus, T; Poehlsen, J; Trunk, U; Mezza, D; Schmidt, B; Kuhn, M; Fromme, R; Sztuk-Dambietz, J; Raab, N; Hauf, S; Silenzi, A; Michelat, T; Xu, C; Danilevski, C; Parenti, A; Mekinda, L; Weinhausen, B; Mills, G; Vagovic, P; Kim, Y; Kirkwood, Henry; Bean, R; Bielecki, J; Stern, S; Giewekemeyer, K; Round, AR; Schulz, J; Dörner, K; Grant, TD; Mariani, V; Barty, A; Mancuso, Adrian; Weierstall, U; Spence, JCH; Chapman, HN; Zatsepin, Nadia; Fromme, P; Kirian, RA; Ros, A

doi:10.26181/21902808.v1

Segmented flow generator for serial crystallography at the European X-ray free electron laser

journal contribution

posted on 2023-01-16, 04:45 authored by A Echelmeier, J Cruz Villarreal, M Messerschmidt, D Kim, JD Coe, D Thifault, S Botha, A Egatz-Gomez, S Gandhi, G Brehm, CE Conrad, DT Hansen, C Madsen, S Bajt, JD Meza-Aguilar, D Oberthür, MO Wiedorn, H Fleckenstein, D Mendez, J Knoška, JM Martin-Garcia, H Hu, S Lisova, A Allahgholi, Y Gevorkov, K Ayyer, S Aplin, HM Ginn, H Graafsma, Andrew J Morgan, D Greiffenberg, A Klujev, T Laurus, J Poehlsen, U Trunk, D Mezza, B Schmidt, M Kuhn, R Fromme, J Sztuk-Dambietz, N Raab, S Hauf, A Silenzi, T Michelat, C Xu, C Danilevski, A Parenti, L Mekinda, B Weinhausen, G Mills, P Vagovic, Y Kim, Henry KirkwoodHenry Kirkwood, R Bean, J Bielecki, S Stern, K Giewekemeyer, AR Round, J Schulz, K Dörner, TD Grant, V Mariani, A Barty, Adrian MancusoAdrian Mancuso, U Weierstall, JCH Spence, HN Chapman, Nadia Zatsepin, P Fromme, RA Kirian, A Ros

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.

Funding

Financial support from the STC Program of the National Science Foundation through BioXFEL under Agreement No. 1231306, NSF ABI Innovations award No. 1565180 and the National Institutes of Health Awards No. R01GM095583, as well as No. 1S10OD021816-01 titled "3D Nanoprinter" is gratefully acknowledged. We also acknowledge European XFEL in Schenefeld, Germany, for provision of X-ray free-electron laser beamtime at Scientific Instrument SPB/SFX (Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography) and would like to thank the instrument group and facility staff for their assistance. In addition, financial support from Cluster of Excellence `Advanced Imaging of Matter' of the Deutsche Forschungsgemeinschaft (DFG)-EXC 2056-project ID 390715994 is gratefully acknowledged. J.C.V. gratefully acknowledges financial support through a CONACYT fellowship. Support from the Australian Research Council through the Centre of Excellence in Advanced Molecular Imaging (CE140100011) is also acknowledged. Please note, Alexander Klujev was previously known as Alexander Klyuev.

History

Publication Date

2020-01-01

Journal

Nature Communications

Volume

11

Issue

1

Article Number

4511

Pagination

10p.

Publisher

Nature Publishing Group

ISSN

2041-1723

Rights Statement

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