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 recorded1-3. 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 Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. 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 processes6. 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.

Accurate determination of segmented X-ray detector geometry

“…Diffraction data can be measured using either X-ray Free Electron Laser [1,2,15,18] or Synchrotron [9,19,20] sources. The common feature of serial crystallography experiments is that a single diffraction pattern is collected from each of a large number of individual crystals in a random orientation.…”

“…This new class of X-ray source produces X-ray pulses of a few tens of femtoseconds duration containing sufficient photons to record the diffraction pattern from individual micron-sized, or smaller, crystals in a single shot [1,2]. Each pulse can measure a different crystal, and diffraction patterns can be measured at the pulse repetition rate, which so far has been up to 120 Hz.…”

High-Coherence Electron and Ion Bunches From Laser-Cooled Atoms

“…In a bid to achieve this goal many different technologies are being developed. These include billion-dollar x-ray free electron lasers which attempt to produce sufficient brightness in an x-ray beam for single-shot imaging of non-crystalline objects [3].…”

Fluid sample injectors for x-ray free electron laser at SACLA

“…By focusing the XFEL pulse to ∼1 µm (∼0.05 µm), the intensity reaches as high as ∼10 18 (∼10 20 ) W cm −2 [3,4] . These characteristics offer research opportunities in various fields of science such as structural biology [5][6][7][8][9] , nonlinear x-ray optics [10,11] , ultrafast physics and chemistry [12][13][14][15] , and high energy density science [16] .…”

“…In the early experiments with XFEL, liquid-jet injectors using a gas dynamic virtual nozzle (GDVN) were applied to protein crystallography [5,7,[20][21][22][23] . This type of injector produced a continuous stream of liquid containing 10 8 -10 9 cm −3 of protein microcrystals [20,21] .…”