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. 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. To further reduce other sources of background, low scattering silicon frames were engineered, as shown in Fig. 1a–c. The silicon frame was designed with robust, efficient and simple sample scanning in mind. The 20-µm diameter holes were an optimal balance between the visibility of holes in the on-axis microscope necessary to align the frame with the X-rays, reduction of the interaction between the wings of the focused beam with the chip, and preventing window membranes from breaking during fabrication. Holey frames were covered with a layer of ultraclean graphene. A fabrication process previously described for smaller free-standing graphene windows33 was modified as depicted in Supplementary Fig. 1 and detailed in Methods. Support frames were tested for graphene cleanliness, coverage, and stability of the graphene upon sample application using low energy electron and light microscopy (Supplementary Fig. 2). The cleanliness of the graphene windows was comparable to that described elsewhere33, 38.

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 Full size image

Silicon frames with freshly prepared ultraclean graphene layers (with and without samples) were glued onto an aluminum frame prior to their introduction into the CXI32, 39 vacuum chamber. Data collection was performed using XFEL pulses of 40 fs duration at 8 keV photon energy and 1.5 mJ pulse energy at the beam focus, giving a calculated peak fluence of about 7 × 1013 photons/µm2. The experimental setup is depicted in Fig. 1a. The frames were scanned at 1.5 s−1 through the XFEL beam. This step scan was performed such that the XFEL pulse intersected every silicon hole, similar to previous fixed target approaches40, 41. Diffraction patterns were collected over two 24-hour shifts.

Preparation of aligned filaments on graphene

We selected Tobacco Mosaic Virus (TMV) fibers as a reference sample, and functional hormone amyloid protofilaments prepared from bombesin and β-endorphin peptides42. TMV has a large asymmetric unit whose 3D structure has been determined to high-resolution by fiber diffraction and refined with cryo-EM6, 43. Soluble bombesin and β-endorphin act both as neurotransmitters in the central nervous system and control a wide spectrum of activities on the cell periphery, and bombesin has putative roles in cancer growth. Both hormones are arranged as amyloid fibrils inside secretory granules of cells42. In contrast to disease-related amyloid fibrils, hormone amyloid fibrils can disassemble into active peptides upon pH change, and they exhibit a very low degree of polymorphism, which is essential to this experiment44. Amyloid fibrils form and maintain their structure under extreme conditions including acidic environments and high temperatures45, and so are not expected to degrade in the vacuum in the XFEL chamber.

A key to obtaining useful diffraction signals from multiple filaments is their mutual alignment. Graphene provides a great benefit in this regard since it exhibits guiding forces to protein filaments, which tend to align them along their fibril axes46, 47. This effect can be clearly observed by comparing images of TMV (Fig. 2a, b), bombesin amyloid fibrils (Fig. 2c, d) and β-endorphin amyloid fibrils (Fig. 2e, f), when they are placed either on amorphous carbon films (Fig. 2a, c, e) or graphene (Fig. 2b, d, f). In contrast to TMV, amyloid fibers are visibly polymorphic and are composed of different numbers of protofibrils (Fig. 2c, e). We observed that individual protofibrils aligned with graphene (Fig. 2d, f), whereas the mature fibers did not.

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 Full size image

Protofibrils were the targets of this experiment, and to initiate their formation we mixed purified peptide solutions of bombesin and β-endorphin with heparin at slightly acid pH values (pH 5.5) mimicking their native acidic environment in secretory granules42. Protofibril growth was imaged by negative-stain transmission electron microscopy (TEM) over four days (see Methods) and the existence of protofibrils was observed between 8–24 h after initiation of filamentation. Fibril suspensions were tested for alignment by depositing droplets on ultraclean graphene sheets dispersed on solid silicon. Atomic force microscopy (AFM) imaging showed that graphene appeared to stop assembly processes and maintain protofibril structures during the time of the AFM measurements (a few hours) (Fig. 2d, f). Protofibril dilution was calibrated to maximize the frequency of single layers.

β-endorphin protofibrils have an average diameter of 3 nm, which was identified from a one-dimensional intensity profile obtained from TEM images of straight fibers44. To estimate the diameter of bombesin protofibrils the signal-to-noise was increased by generating seven 2D class averages of fibers from the TEM micrographs (Supplementary Fig. 3). Pixel intensities in columns parallel to the fiber axis were summed to a one-dimensional profile, which revealed diameters of bombesin fibers ranging from 8.8 to 11.3 nm. Modulations in these intensity profiles (Supplementary Fig. 3b) suggest that bombesin fibers are composed of three to four protofibrils each with a width of 2–3 nm. Imaging TMV in the same micrograph shows that its diameter is about six times larger than that of the individual bombesin protofibrils (Fig. 2c, Supplementary Fig. 3).

Scattering intensities of fibril and background components

A total of 126,768 diffraction frames were acquired from four dilutions of all three fiber types, on empty holes and holes covered with only graphene and no sample. 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).

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 Full size image

Frames showing diffraction from graphene-covered sample-free holes were selected from a series of 4352 detector frames with diluted TMV. The selection criteria are described in Supplementary Fig. 5 and Methods. The average background from sample-free holes contains about 119,556 scattered photons (Fig. 3a, Supplementary Fig. 4a). This is equivalent to about 0.05 photons/pixel. The scattering from empty holes was determined from a series of 1569 frames. Empty holes give rise to measurable diffuse scattering from the silicon chip (Supplementary Fig. 4b). The average total background per image from the series exposing only empty holes, excluding beam-off events, was about 101,345 scattered photons.

We find that scattering from empty holes is similar to that of graphene-covered sample-free holes, indicating that the main component of the average background (Fig. 3a, Supplementary Fig. 4a) is due to the empty holes alone. Additional background may contain contributions from misclassified very weak TMV hits (the fraction of patterns containing sample diffraction), as well as the graphene layer. Other sources of background seen in the difference (Supplementary Fig. 4c) between the average background (Fig. 3, Supplementary Fig. 4a) and the background from empty holes (Supplementary Fig. 4b) may be attributed to the parasitic scattering of the XFEL and iron fluorescence in the steel vacuum chamber48.

Hit fractions of 30–50% were achieved with samples that were diluted 20–250 times, starting with initial peptide concentrations of 1 mg/ml. Diffraction patterns from TMV exhibited layer lines in one or more orientations, indicating the presence of single or multiple layers of protofibrils in the nanofocus. An example diffraction frame from TMV with a single orientation on the graphene is shown in Fig. 3b. A pattern from bombesin is shown in Fig. 3c. Radial sections of these patterns are plotted in Fig. 3d. The signal from the amyloid and TMV are seen to extend to 2.4 Å and 2.7 Å, respectively. The background contribution from free-standing graphene is seen to be two orders of magnitude lower than the sample signal in Fig. 3d.

Diffraction by TMV fibers

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 XFEL pattern resembles the azimuthally averaged synchrotron X-ray diffraction pattern. The qualitative agreement between the strong features suggests that the structure is not damaged in vacuum relative to the solvated form up to relatively high resolution. We selected 37 TMV XFEL frames with well-defined layer lines similar to Fig. 4a and calculated the period of the molecular structure along the c axis (fiber axis) from the layer line spacing. 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.

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 publication6, 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 Full size image

Asymmetric features in single-shot XFEL diffraction patterns

A conventional fiber specimen contains many molecules with random axial rotations, and random directions either parallel or antiparallel to the fiber axis. A conventional fiber diffraction pattern is, therefore, cylindrically averaged, and so is symmetric about the equator (horizontal axis) and the meridian (vertical axis), as is evident in Fig. 4b. However, in some of the XFEL diffraction patterns, such as Fig. 4a, there is evidence that this symmetry is not present and there are observable differences along layer lines to the left and right of the meridian. Intensity profiles of the left and right halves of some of the layer lines are compared in Fig. 4c. The asymmetry indicates that the XFEL diffraction patterns from TMV are not cylindrically averaged, and that protofibrils with only one or a few axial rotations may be simultaneously exposed in the XFEL focus. Such patterns potentially contain more information than the cylindrically averaged patterns measured in conventional fiber diffraction experiments50.

Although the number of protofibrils within the focus is limited, their exact number is difficult to determine. The number of protofibrils was estimated from examination of tapping-mode AFM images of graphene-covered silicon next to the selected windows (similar to Fig. 2a–c) as the free-standing graphene is too fragile to withstand AFM analysis. Based on this analysis and the ~150 nm XFEL focal diameter, we estimate that fewer than about 50 amyloid protofibrils and about eight TMV fibers contributed to the single diffraction patterns shown in Fig. 3.

Diffraction by amyloid protofibrils

Amyloid protofibrils are about six times smaller in diameter than TMV particles (Fig. 2b and S3), and therefore exhibit broader diffraction features. Single diffraction snapshots from amyloid protofibrils of bombesin and β-endorphin are shown in Fig. 5a, b. These patterns exhibit strong intensity on the equator and a strong meridional layer line at about 4.8 Å due to the characteristic spacing of β-strands in β-sheets typical for amyloids10. This preserved c-repeat indicates that there are no global structural changes due to the experimental conditions. The second layer line at ~2.4 Å (4.8 Å / 2) on the meridian is also present in single snapshots.

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 Full size image

Forty diffraction frames from each amyloid data set (bombesin and β-endorphin) with well-defined layer lines similar to Fig. 5a, b were selected manually, as existing hit-finding methods were found to be not suitable for detecting layer lines in the somewhat diffuse patterns of this kind. Patterns were aligned and registered in reciprocal space after their rotation angle around the beam axis (φ) and the deviation of the fiber axis from the normal to the beam axis (β) (Supplementary Fig. 7) were determined. The tilt angle between the fiber axis and the X-ray beam varied within a small range, independent of the substrate tilt due to buckling of graphene across the holes. The oriented frames were then mapped into reciprocal space (R, Z) for subsequent analysis, with coordinates normal (R), and parallel (Z), to the fiber axis51. The mapped patterns were merged, symmetrized and background-subtracted to give an averaged pattern in (R, Z) space with an improved signal-to-noise ratio (Fig. 5c, d). Averaged equatorial intensity profiles shown in Fig. 5e, f were used to determine positions of the equatorial maxima. For bombesin, three peaks including one pronounced equatorial peak at 10.6 Å are discernible (Figure 5e). β-endorphin fibrils show five peaks amongst which there are three pronounced maxima at 8.1 Å, 9.9 Å, and 12.3 Å labeled 3, 4, and 5, respectively (Fig. 5f). Both equatorial and meridional peaks are summarized in Table 1.