Femtosecond X-ray diffraction from two-dimensional protein crystals.

Matthias Frank,Bill Pedrini,Nadia A Zatsepin,Celestino Padeste,James E Evans,Mark S Hunter,Richard A Kirian,W Henry Benner,Stephen M Lane,Anton Barty,Garth J Williams,Kaiqin Chu,Marc Messerschmidt,John C H Spence,Brent W Segelke ,Alexander Graf ,T Pardini ,Ching‐Ju Tsai ,Gebhard F X Schertler ,Stefan P Hau‐Riege ,Matthew A Coleman ,Xiaodan Li ,Donald E Carlson ,M Seibert ,Sébastien Boutet

doi:10.1107/s2052252514001444

Abstract

X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

Highlights

X-ray crystallography has been the leading method for atomic resolution structure determination of biological macromolecules since the 1950s (RCSB, 2013), yet this technique is typically limited to macroscopic three-dimensional (3-D) protein crystals larger than 10 mm per side (Holton & Frankel, 2010) when using synchrotron light sources
The extremely short femtosecond-duration pulses delivered by X-ray free-electron laser (XFEL) have a peak brightness many orders of magnitude greater than synchrotron sources permitting the collection of X-ray diffraction from even smaller samples including 2-D crystals (Kewish et al, 2010) and single particles (Neutze et al, 2000) at doses significantly exceeding the normal tolerable room-temperature radiation dose (Redecke et al, 2013)
Transmission X-ray diffraction patterns from individual sugarembedded 2-D protein crystals highlight the potential for XFELs to enable the study of 2-D crystals of biological macromolecules including undamaged membrane proteins and soluble proteins at room temperature

Summary

Introduction

X-ray crystallography has been the leading method for atomic resolution structure determination of biological macromolecules since the 1950s (RCSB, 2013), yet this technique is typically limited to macroscopic three-dimensional (3-D) protein crystals larger than 10 mm per side (Holton & Frankel, 2010) when using synchrotron light sources. While transmission electron microscopy has yielded protein doi:10.1107/S2052252514001444 95 research papers structures from 2-D crystals of both soluble proteins (Nogales et al, 1998) and membrane proteins (Gonen et al, 2005; Henderson et al, 1990), fewer than 30 unique structures have been solved to better than 4 Awith this technique. For each of these above-mentioned methods, achieving high-resolution structures from 2-D crystals can be significantly hindered by radiation damage. 2-D crystals possess a compact support along the beam direction which presents entirely new possibilities for solving the phase problem iteratively in 3-D (Spence et al, 2003)

Methods

Results

Conclusion