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

Austin Echelmeier,Grant Mills,Stephan Stern,J Domingo Meza-Aguilar,Katerina Dörner,Dominik Oberthür,Saša Bajt,John C H Spence,S Aplin ,Henry Kirkwood,Chen Xu,Debra T Hansen,Derek Mendez,Daihyun Kim,A Parenti ,H Ginn ,Joachim Schulz,A Silenzi ,Britta Weinhausen,A Klujev ,Caleb Madsen,Chelsie E Conrad,Kartik Ayyer,Jorvani Cruz Villarreal,Adam Round ,Natascha Raab,Henry N Chapman,H Fleckenstein ,Cyril Danilevski,J Poehlsen ,Y Gevorkov ,Sabine Botha,Klaus Giewekemeyer,H Graafsma ,Gerrit Brehm,Richard A Kirian,Richard Bean,J Sztuk-Dambietz ,Patrik Vagovič ,Yoonhee Kim,Anton Barty,Jesse Coe ,Andrew J Morgan,Raimund Fromme,Manuela Kuhn,Hao Hu,Darren Thifault,U Trunk ,J Knoška ,Torsten Laurus,Petra Fromme,Thomas D Grant,Sahir Gandhi,Léonce Mekinda ,Valerio Mariani,Marc Messerschmidt,Adrian P Mancuso,Max O Wiedorn,D Greiffenberg ,José M Martín-García ,Uwe Weierstall,Bernd Schmidt,Nadia A Zatsepin ,Stella Lisova,Thomas Michelat,Ana Egatz-Gómez ,D Mezza ,Johan Bielecki,Steffen Hauf,A Allahgholi ,Alexandra Ros

doi:10.1038/s41467-020-18156-7

Abstract

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.

Highlights

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography
The current Acoustic droplet ejectors (ADEs) realizations are incompatible with the short MHz spacing of pulses at the European XFEL (EuXFEL), as they have been demonstrated at orders of magnitude lower frequency at available XFELs to date[10,34,35,36]
We adapted this approach for a workflow compatible with the early user experiments at the EuXFEL as depicted in Fig. 1a, which allows for macromolecular structural studies at XFELs at room temperature in a vacuum chamber

Summary

Introduction

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. During the beam time experiment, we investigated the influence of the aqueous and oil flow rates on the crystal hit fraction, defined here as the average number of crystal diffraction patterns per X-ray pulse.

Results

Conclusion