Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

Carolin Seuring,Mark S Hunter,Sébastien Boutet,Katerina Dörner,Miriam Barthelmeß ,Matthias Frank,H Ginn ,David H Wojtas,Anton Barty,Jean Nicolas Longchamp ,Annette Eva Langkilde ,Antoine Loquet,Mao Liang ,Henning Stahlberg,Rick P Millane,T Pardini ,Kartik Ayyer,Silje Skeide Fuglerud ,Jason E Koglin ,A Meents ,Matthew A Coleman,Eleftheria Filippaki,Birgit Habenstein,Philippe Ringler,Greger Hammarin,Roland Riek,Henry N Chapman

doi:10.1038/s41467-018-04116-9

Abstract

Here we present a new approach to diffraction imaging of amyloid fibrils, combining a free-standing graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 Å and 2.4 Å resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects.

Highlights

Background dtobacco mosaic virus (TMV) 0 Bombesin PhotonsBombesin TMV Background R (Å–1)b l = 9 l = 6 l = 3E l = –3 l = –6 l = –9 L c L + R + M l = ± 3R l = ± 6 l = ± 9
Experiments were conducted in vacuum to minimize background scattering from air, and the X-ray beam was focused to a spot size of about 150 nm full-width at half maximum (FWHM) to maximize the flux incident on individual protofibrils
By naturally aligning TMV filaments and protofibrils composed of bombesin and β-endorphin peptides, graphene fixes the alignment of the molecule during exposure

Summary

Results

XFEL imaging of fibrils on free-standing graphene windows. We used an ultraclean graphene layer placed on a holey silicon support frame to deliver non-crystalline filaments into the XFEL beam focus. To compare the scattering intensity from the fibril and graphene components and for calibrating background subtraction, we characterized the X-ray scattering from graphene-covered holes (Fig. 3) and sample-free, empty holes (Supplementary Fig. 4). To demonstrate the structural integrity of the samples under our experimental conditions, we compare a single XFEL diffraction pattern from TMV exhibiting 24 layer lines (Fig. 4a) to a synchrotron diffraction pattern obtained from a specimen containing millions of TMV filaments aligned in well-oriented gels (Fig. 4b)[6]. The average value is 68.8 Å, which agrees with the known value of 68.7 Å43, 49, and the values from individual patterns are equal to this value within the error bars (Supplementary Fig. 6) This suggests that the global structure of TMV is maintained during the experiment.

Background d

Discussion

Methods