Abstract
Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.
Highlights
Using pulses from X-ray free-electron lasers (XFEL) with sufficiently short duration to outrun radiation damage, the structures of biological macromolecules can be obtained from nanocrystals at room temperature[1,2]
Using a viscous matrix such as lipidic cubic phase (LCP), sample consumption can be reduced to about 100 nl/min or less[9]
Low sample consumption can be achieved with nanoflow electrospinning injection using a microfluidic electro-kinetic sample holder (MESH), which requires the sample to be mixed into a cryo-protective buffer[10]
Summary
Our DFFN system (Fig. 1) reduces sample consumption up to eight fold compared to a GDVN, can inject crystals in their native crystallization buffers, and is suitable for time-resolved mixing experiments and future high repetition rate X-ray sources such as LCLS-II. For CpGV, the flow-rate of 3–5 μl/min produced an inner jet width matched both to the size of the X-ray beam and that of the crystals Either the use of water or ethanol as sheath liquids leads to stable jets at much reduced sample flow-rates as compared to normal GDVN injection. A new injection system for SFX experiments, referred to as a double-flow focus nozzle (DFFN), enables structural biology experiments at room temperature on samples of high biological importance as demonstrated by the first structures of RNA polymerase II and HPCD determined at non-cryogenic temperatures.
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