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

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.

Fig. 1

Experimental overview. a Silicon chips covered with a monolayer of graphene and a layer of fibrils were mounted in vacuum at the CXI beamline at LCLS and scanned through the XFEL focus. b Fixed-targets were made of a square silicon frame patterned with an array of 12 × 13 windows. Scalebar is 5 mm. c A single window contained 81 holes of either 20 or 30 μm in diameter arrayed on a hexagonal lattice, resulting in 12,636 holes over a 2.54 × 2.54 cm area. Scalebar is 100 μm. d An atomic force microscope image of a hole covered with graphene and fibrils is shown. Fibrils were imaged next to the hole on the silicon frame, as tapping without the support destroys the graphene layer. Scalebar is 20 nm

Fig. 2

Preparation of TMV filaments and amyloid protofibrils on graphene. a, b Representative negative-stain TEM and AFM images of TMV, c–d bombesin filaments, and e, f β-endorphin filaments are shown. Negative-stain images (a, c, e) were acquired on fibrils placed on amorphous carbon films and AFM images (b, d, f) on graphene. Scalebars are 100 nm. b TMV fibrils align naturally on graphene over hundreds of nanometers. However, on the micrometer scale, aligned and randomly ordered fibrils are co-present. c Bombesin protofibrils associate laterally to form fibers, which randomly twist. A single preparation may consist of different polymorphs, e.g., twisted fibers and fibril rafts which are depicted here with arrows and squares, respectively. Bombesin fibers were mixed with TMV to compare their thickness. d The alignment of bombesin protofibrils on graphene is shown. Mature fibers are detected at larger magnifications. e β–endorphin protofibrils associate laterally to form twisted and striated fibers. f Aligned β–endorphin protofibrils were observed on graphene supports. To confirm that the features that are being imaged by the AFM are from the sample and not an artifact caused by the probe, the sample was rotated by 30° with respect to the scanning direction. Dashed circles represent the XFEL focus with FWHM = 150 nm

Fig. 3

Diffraction images obtained at the LCLS in the CXI nanofocus chamber. a The average background from 1,607 selected frames with graphene but without sample. The diffuse scattering of the silicon and some contamination is visible. Single frames from b TMV and c the amyloid bombesin. a–c The grayscale shows photons per pixel. The average background contains 119,556 scattered photons, which is equivalent to about 0.050 photons/pixel (a). d Traces from the diagonal lines in (a–c) plotted as a function of reciprocal resolution R (Å⁻¹). The average background of graphene-covered holes is two orders of magnitude lower than that due to the samples

Fig. 4

Comparison of XFEL and conventional X-ray fiber TMV diffraction patterns. a A single XFEL snapshot of TMV protofibrils on graphene is shown. The resolution at the center-edge is 3.86 Å. Left and Right layer lines, equatorial and meridional axes are labeled L, R, E, and M, respectively. b A classical X-ray fiber diffraction pattern from millions of mutually aligned TMV fibrils. Reprinted from publication, Copyright (1989), with permission from Elsevier. c Magnifications of three symmetry related layer lines (l = ± 3, l = ± 6, l = ± 9) are shown as a function of resolution R. The left and right sides of the layer lines are indicated with L and R, and positive and negative layer lines with + and −, respectively. The left layer lines are flipped along the vertical axis to match the profile of the right layer lines

Fig. 5

XFEL Diffraction patterns obtained from amyloid fibrils. Fibrils composed of bombesin and β-endorphin are shown on the left and right, respectively. a–b Single diffraction snapshots from aligned protofibrils, and background-subtracted merged patterns obtained from 40 diffraction snapshots each of c bombesin and d β-endorphin are shown. e, f Averaged intensity profiles as a function of reciprocal resolution over a band of width eight pixels ((e) bombesin) and 22 pixels ((f) β-endorphin) centered on the equator. Peaks in the equatorial profiles are marked. All peaks are summarized in Table 1

Fig. 6

Comparison of conventional X-ray patterns to merged XFEL patterns. Diffraction patterns from amyloid fibers composed of a Aβ(1–42), b IAPP(1–37), c Aβ(11–25), and d Het-s(218-289) are shown. The equator and the most prominent layer lines are marked on the right side. The white and black arrow mark the meridional reflection at about 4.8 Å and the equatorial reflection at about ~10 Å characteristic for stacked β-sheets and present in (a–d), respectively. Note, that all amyloid fibrils are non-crystalline except for (c), which is crystalline. Merged XFEL diffraction patterns of bombesin (e) and β-endorphin fibrils (f) extending to 2.4 Å resolution are shown for qualitative comparison. a–c are reprinted from publication, Copyright (2010), with permission from Elsevier. d is reprinted from publication (https://pubs.acs.org/doi/abs/10.1021%2Fbi5002807). Further permissions related to the material excerpted should be directed to the ACS