High-resolution protein structure determination by serial femtosecond crystallography

Abstract

Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

Fig. 1

Experimental geometry for SFX at the CXI instrument. Single-pulse diffraction patterns from single crystals flowing in a liquid jet are recorded on a CSPAD at the 120-Hz repetition rate of LCLS. Each pulse was focused at the interaction point by using 9.4-keV x-rays. The sample-to-detector distance (z) was 93 mm.

Fig. 2

(A) Final, refined 2mF_obs − DF_calc (1.5σ) electron density map (17) of lysozyme at 1.9 Å resolution calculated from 40-fs pulse data. (B) F_obs(40 fs) − F_obs (synchrotron) difference Fourier map, contoured at +3 σ (green) and −3 σ (red). No interpretable features are apparent. The synchrotron data set was collected with a radiation dose of 24 kGy.