Additional x-ray diffraction analyses. Raster-scans of CT domain (a) and 4RepCT (b) silk fibers. Composite image of CCD-patterns (‘pixels’) obtained from the raster-scans. Note that the scale of all composite images is the same. The X-ray diffraction pattern of aggregated 4Rep: individual diffraction pattern (c) and azimuthally averaged pattern (c).



METHODS

Synchrotron radiation (SR) experiments were performed at the ESRF-ID13 beamline using a monochromatic beam of λ = 0.09996 nm75. The beam was focused by crossed mirrors to an 0.7x0.7 (μm)2 spot at the sample position with a flux of 5x10^10 photons/s. A Frelon CCD detector76 with 100 mm converter screen, 2Kx2K pixels of 51x51 (μm)2 size and 16 bit readout was used. The detector was 2x2 times binned. A short section was cut by microscissors from a piece of fiber and attached by fast glue to a tapered glass capillary, with the fiber vertically oriented. The capillary was fixed by a magnetic support piece to the beamline scanning goniometer. The fiber section was kept during diffraction experiments at 100 K by an Oxford Cryosystems nitrogen cryoflow system. No icing was observed during the experimental period. The sample-to-detector distance was determined as 76.3 mm using an Al2O3 calibration sample. In order to improve the counting statistics, the diffraction patterns were averaged over several patterns measured during a horizontal scan across the fiber. The corresponding averaged background patterns were subtracted. Data collection was done by 2D raster-scans using 5 or 10 μm steps. After every raster-step a WAXS-pattern was collected. A typical collection time for a diffraction pattern was 1 s. The scattering measured during a raster-scan outside of the fiber was subtracted as background. The FIT2D program was used for data analysis and data display77. 

The crystallinity index (Xc) is approximated as:

Xc =   (sum(I))/((sum(I)+ sum(B)))  𝑥100		Eq. 1

Where is sum(I) the sum of Bragg peak intensities and sum(B) the diffuse background scattering.

For the SR experiment, a slightly different CT construct was used, see supporting information Figure S2 and S16. Therefore, complementary X-ray experiments were carried out for fibers from the current construct, using a Cu Kα X-ray source, λ=1.5418 Å. An Eiger R 1M Horizontal detector was used and the sample-to-detector distance was 110.8 mm. Experiments were conducted with an acquisition time of 10 min with 3 frames for 4RepCT and 18 frames for the CT domain. The obtained diffraction patterns confirmed that no major differences can be observed between the two CT constructs (Figure S16).



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