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Raw data of scanning electron microscopy (SEM), atomic force microscopy (AFM), force spectroscopy, data analysis and plotting, optical microscopy, and finite element simulations (FEA) for our manuscript. File Formats AFM raw data is provided in Gwyddion format, which can be viewed using the Gwyddion AFM viewer, which has been released under the GNU public software licence GPLv3 and can be downloaded free of charge at http://gwyddion.net/ Optical microscopy data is provided in JPEG format SEM raw data is provided in TIFF format Data analysis codes were written in MATLAB (https://www.mathworks.com/products/matlab) and stored as *.m files Imported raw data to MATLAB and saved MATLAB data were stored as MATLAB multidimensional arrays (MATLAB “struct” data format, *.mat files) FEA results were saved as text files, .txt files) Data (Folder Structure) The data in the dataverse is best viewed in Tree mode. Read me file.docx More Explanations of analysis in docx format. Figure 1 Figure 1 - panel b.jpg (5.5 MB) Optical micrograph (JPEG format) Figure 1 - panel c - AFM Raw Data.gwy (8.0 MB) AFM raw data (Gwyddion format) Figure 1 - panel e - P0_Force-curve_raw_data.txt (3 KB) Raw force-displacement data at P0 (text format) Figure 1 - panel e - Px_Force-curve_raw_data.txt (3 KB) Raw force-displacement data at Px (text format) Figure 1 - panel e - Py_Force-curve_raw_data.txt (3 KB) Raw force-displacement data at Py (text format) Figure 1 - panel e - P0_simulation_raw_data.txt (12 KB) FEA simulated force-distance data at P0 (text format) Figure 1 - panel e - Px_simulation_raw_data.txt (12 KB) FEA simulated force-distance data at Px (text format) Figure 1 - panel e - Py_simulation_raw_data.txt (12 KB) FEA simulated force-distance data at Py (text format) Figure 1 - panel e - FCfindc.m (2 KB) MATLAB code to calculate inverse optical lever sensitivity (InverseOLS) of AFM cantelever (matlab .m format) Figure 1 - panel e - FreqFindANoise_new.m (2 KB) MATLAB code to calculate white noise constant, A (Explained in the Read me file) (matlab .m format) Figure 1 - panel e - FreqFindQ_new.m (4 KB) MATLAB code to calculate Q factor of the AFM cantelever (matlab .m format) Figure 1 - panel e - FCkeff.m (2 KB) MATLAB code to calculate the effective spring constant k of the AFM cantelever (matlab .m format) Figure 1 - panel e - FCimport.m (7 KB) MATLAB code to import raw force-displacement data into MATLAB (matlab .m format) Figure 1 - panel e - FCForceDist.m (2 KB) MATLAB code to convert raw force-displacement data into force-distance data (matlab .m format) Figure 1 - panel e - Figure 1- Panel e - data.mat (6 KB) MATALB struct data file for calibrated force-distance data at all indentation points (matlab .mat format) Figure 1 - panel e - Panel_e_MatlabCode.m (6 KB) MATALB code for plotting experimental and simulated force curves in panel e (matlab .m format) Figure 1 - panel e - Read me file - force curve calibration.docx (14 KB) Explains force curve calibration (.docx format) Figure 1 - panel e - Read me file - lever spring constant calibration.docx (14 KB) Explains AFM lever spring constant calibration (.docx format) Figure 2 Figure 2 - panel a - MATLAB data.mat (2.6 KB) MATALB data file for simulated data (matlab .mat format) Figure 2 - panel b - MATLAB data.mat (2.4 KB) MATALB data file for simulated data (matlab .mat format) Figure 2 - panel a - simulation raw data.txt (5.0 KB) Raw simulation data: xyz coordinates of the nodes of deformed FEA mesh (text format) Figure 2 - panel b - simulation raw data.txt (5.0 KB) Raw simulation data: xyz coordinates of the nodes of deformed FEA mesh (text format) Figure 2 - panel ab - MATLABcode.m (1.0 KB) MATALB code for plotting panel a b figures (matlab .m format) Figure 2 - panel c - Degree of Anisotropy datacode.m (1.0 KB) MATALB code for plotting panel c graph (matlab .m format) Figure 3 Figure 3 - panel a - App_curve_1_raw_data.txt (35 KB) Raw force-displacement data approach curve 1 (text format) Figure 3 - panel a - App_curve_2_raw_data.txt (34 KB) Raw force-displacement data approach curve 2 (text format) Figure 3 - panel a - App_curve_3_raw_data.txt (34 KB) Raw force-displacement data approach curve 3 (text format) Figure 3 - panel a - App_curve_4_raw_data.txt (34 KB) Raw force-displacement data approach curve 4 (text format) Figure 3 - panel a - Ret_curve_1_raw_data.txt (35 KB) Raw force-displacement data of retract curve 1 (text format) Figure 3 - panel a - Ret_curve_2_raw_data.txt (35 KB) Raw force-displacement data of retract curve 2 (text format) Figure 3 - panel a - Ret_curve_3_raw_data.txt (35 KB) Raw force-displacement data of retract curve 3 (text format) Figure 3 - panel a - Simulation_raw_data-part 1.txt (43 KB) simulated force-displacement data of -part 1 (text format) Figure 3 - panel a - Simulation_raw_data-part 2.txt (43 KB) simulated force-displacement data of -part 2 (text format) Figure 3 - panel a - FCfindc.m (2 KB) MATLAB code to calculate inverse optical lever sensitivity (InverseOLS) of AFM cantelever (matlab .m format) Figure 3 - panel a - FreqFindANoise_new.m (2 KB) MATLAB code to calculate white noise constant, A (Explained in the Read me file) (matlab .m format) Figure 3 - panel a - FreqFindQ_new.m (4 KB) MATLAB code to calculate Q factor of the AFM cantelever (matlab .m format) Figure 3 - panel a - FCkeff.m (2 KB) MATLAB code to calculate the effective spring constant k of the AFM cantelever (matlab .m format) Figure 3 - panel a - FCimport.m (7 KB) MATLAB code to import raw force-displacement data into MATLAB (matlab .m format) Figure 3 - panel a - FCForceDist.m (2 KB) MATLAB code to convert raw force-displacement data into force-distance data (matlab .m format) Figure 1 - panel e - Read me file - force curve calibration.docx (14 KB) Explains force curve calibration (.docx format) Figure 1 - panel e - Read me file - lever spring constant calibration.docx (14 KB) Explains AFM lever spring constant calibration (.docx format) Figure 3 - panel b - SEM Raw Data.tiff (9 KB) SEM raw image of broken silk membrane due to extreme indentation (.tiff format) 

Raw data of optical microscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), and diameter measurements of the exfoliated and self-assembled nanofibrils for our manuscript. File Formats AFM raw data is provided in Gwyddion format, which can be viewed using the Gwyddion AFM viewer, which has been released under the GNU public software licence GPLv3 and can be downloaded free of charge at http://gwyddion.net/ Optical microscopy data is provided in JPEG format SEM raw data is provided in TIFF format Data analysis codes were written in MATLAB (https://www.mathworks.com/products/matlab) and stored as *.m files Data analysis results were stored as MATLAB multidimensional arrays (MATLAB “struct” data format, *.mat files) Data (Folder Structure) The data in the dataverse is best viewed in Tree mode. ReadMe.md This description in Markdown format. Figure 2 - Microscopy Raw Data Figure 2 - panel a.jpg (7.2 MB) Optical micrograph (JPEG format) Figure 2 - panel b.jpg (6.1 MB) Optical micrograph (JPEG format) Figure 2 - panel c f.tif (1.2 MB) SEM raw data (TIFF format) Figure 2 - panel d.tif (1.2 MB) SEM raw data (TIFF format) Figure 2 - panel e - Exfoliated Fibrils.gwy (32.0 MB) AFM raw data (Gwyddion format) Figure 3 - AFM Raw Data Figure 3 - Panel a - Exfoliated fibrils.gwy (81.5 MB) AFM raw data (Gwyddion format) Figure 3 - Panel c - Self-assembled fibrils.gwy (24.0 MB) AFM raw data (Gwyddion format) Figure 3 - Diameter Measurements Figure 3a and Figure 3c show the AFM images of exfoliated and self-assembled nanofibrils, respectively. However, due to the AFM tip-induced broadening of lateral dimensions of small features (such as nanofibrils), the diameters of nanofibrils are generally overestimated in AFM images. Hence, the diameters of the nanofibrils were estimated as the full width at half maximum (FWHM) value of line scans taken over nanofibrils perpendicular to their axial direction. Line profiles were taken at multiple locations using Gwyddion, and the raw data were stored in MATLAB struct files (lineProfileData_Exfoliated.mat and lineProfileData_Self-Assembled.mat). These data files can be directly imported into MATLAB and will appear as “DataExf” and “DataSA” in MATLAB workspace. For instance, “DataExf.x{i}” contains the x-axis data of i-th line profile, and “DataExf.y{i}” contains the y-axis data of i-th line profile. The MATLAB codes MainCode_Exf.m and MainCode_SA.m are used to fit Gaussian curves for each line profile and calculate the FWHM. The *.m files for functions gaussian.m and createFit.m must be in the same folder as the file for the main code. The main code generates figures for each line profile containing raw line profile, related Gaussian fit, and FWHM. These FWHM values are considered as the diameters of the fibrils and stored in variables called “Exf_Dia” and “SA_Dia”. Finally, these values are plotted in a histogram and calculate the statistics such as the mean and the standard deviation. Exfoliated createFit.m (1.1 KB) MATLAB code file (see above) gaussian.m (134 B) MATLAB code file (see above) lineProfileData_Exfoliated.mat (11.7 KB) Line profiles for exfoliated nanofibrils (MATLAB struct format) MainCode_Exf.m (1.8 KB) MATLAB code file (see above) Line profile raw data - Exfoliated Folder with all corresponding cross section raw data in ASCII format Self Assembled createFit.m (1.1 KB) MATLAB code file (see above) gaussian.m (134 B) MATLAB code file (see above) lineProfileData_Self-Assembled.mat (9.9 KB) Line profiles for self-assembled nanofibrils (MATLAB struct format) MainCode_SA.m (1.8 KB) MATLAB code file (see above) Line profile raw data - SelfAssembled Folder with all corresponding cross section raw data in ASCII format Figure 4 - AFM Raw Data Figure 4 - Panal a.gwy (73.4 MB) AFM raw data (Gwyddion format) Figure 4 - Panel e.gwy (42.0 MB) AFM raw data (Gwyddion format) 

Biomaterials with outstanding mechanical properties, including spider silk, wood, and cartilage, often feature an oriented nanofibrillar structure. The orientation of nanofibrils gives rise to a significant mechanical anisotropy, which is extremely challenging to characterize, especially for microscopically small or inhomogeneous samples. Here, a technique utilizing atomic force microscope indentation at multiple points combined with finite element analysis to sample the mechanical anisotropy of a thin film in a microscopically small area is reported. The system studied here is the tape‐like silk of the Chilean recluse spider, which entirely consists of strictly oriented nanofibrils giving rise to a large mechanical anisotropy. The most detailed directional nanoscale structure–property characterization of spider silk to date is presented, revealing the tensile and transverse elastic moduli as 9 and 1 GPa, respectively, and the binding strength between silk nanofibrils as 159±13 MPa. Furthermore, based on this binding strength, the nanofibrils’ surface energy is derived as 37 mJ m⁻², and concludes that van der Waals forces play a decisive role in interfibrillar binding. Due to its versatility, this technique has many potential applications, including early disease diagnostics, as underlying pathological conditions can alter the local mechanical properties of tissues.