Fixed-target protein serial microcrystallography with an x-ray free electron laser

Abstract

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Figure 1. Experimental overview.

Protein crystals embedded in Paratone-N were placed on fixed-targets and measured in vacuo at the Coherent X-ray Imaging (CXI) beam line of the LCLS.

Figure 2. Typical single diffraction pattern from a REP24 crystal preserved in Paratone-N using the CXI instrument of the LCLS.

The diffraction pattern contains Bragg peaks to 2.5 Å resolution (2Å resolution is indicated by the white circle) and was indexed with unit cell parameters that were consistent with the macrocrystal unit cell of REP24.