Femtosecond X-ray protein nanocrystallography

Abstract

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

Figure 1. Femtosecond nanocrystallography

Nanocrystals flow in their buffer solution in a gas-focused, 4-μm-diameter jet at a velocity of 10 m s⁻¹ perpendicular to the pulsed X-ray FEL beam that is focused on the jet. Inset, environmental scanning electron micrograph of the nozzle, flowing jet and focusing gas. Two pairs of high-frame-rate pnCCD detectors record low-and high-angle diffraction from single X-ray FEL pulses, at the FEL repetition rate of 30 Hz. Crystals arrive at random times and orientations in the beam, and the probability of hitting one is proportional to the crystal concentration.

Figure 2. Coherent crystal diffraction

Low-angle diffraction patterns recorded on the rear pnCCDs, revealing coherent diffraction from the structure of the photosystem I nanocrystals, shown using a logarithmic, false-colour scale. The Miller indices of the peaks in a were identified from the corresponding high-angle pattern. In c we count seven fringes in the b* direction, corresponding to nine unit cells, or 250 nm. Insets, real-space images of the nanocrystal, determined by phase retrieval (using the Shrinkwrap algorithm) of the circled coherent Bragg shape transform.

Figure 3. Diffraction intensities and electron density of photosystem I

a, Diffraction pattern recorded on the front pnCCDs with a single 70-fs pulse after background subtraction and correction of saturated pixels. Some peaks are labelled with their Miller indices. The resolution in the lower detector corner is 8.5 Å. b, Precession-style pattern of the [001] zone for photosystem I, obtained from merging femtosecond nanocrystal data from over 15,000 nanocrystal patterns, displayed on the linear colour scale shown on the right. c, d, Region of the 2mF_o − DF_c electron density map at 1.0σ (purple mesh), calculated from the 70-fs data (c) and from conventional synchrotron data truncated at a resolution of 8.5 Å and collected at a temperature of 100 K (d) (Methods). The refined model is depicted in yellow.

Figure 4. Pulse-duration dependence of diffraction intensities

Plot of the integrated Bragg intensities of photosystem I nanocrystal diffraction as a function of photon momentum transfer, q = (4π/λ)sin(θ) = 2π/d (wavelength, λ; scattering angle 2θ; resolution, d) for pulse durations of 10, 70 and 200 fs. Averages were obtained by isolating Bragg spots from 97,883, 805,311 and 66,063 patterns, respectively, normalized to pulse fluence. The error in each plot is indicated by the thickness of the line. The decrease in irradiance for 200-fs pulses and d < 25 Å indicates radiation damage for these long pulses, which is not apparent for 70-fs pulses and shorter.