Annexin V assembly dynamics on lipid bilayers by High-Speed AFM

This dataset contains curated high-speed atomic force microscopy (HS-AFM) videos capturing the dynamic assembly of Annexin V on supported lipid bilayers. Each movie is accompanied by a metadata table detailing key acquisition parameters and imaging conditions.This dataset is released as an open resource and is made freely available to support reuse in high-speed AFM data analysis, algorithm development, machine learning applications, and studies of membrane protein assembly dynamics.Methods SummaryMembrane: DOPC:DOPS = 8:2Protein: Annexin V (33 kDa, human placenta) in imaging solutionImaging Buffer: 10 mM HEPES (pH 7.4), 150 mM NaCl, 2 mMCaCl₂AFM: NanoRacer (Bruker)Probe: USC-F1.2-k0.15 (NanoWorld) Full method description Annexin V (33 kDa, human placenta) was purchased from Sigma-Aldrich, and lipids dioleoyl-phosphatidylcholine (DOPC) and dioleoyl-phosphatidylserine (DOPS) were obtained from Avanti Polar Lipids. Lipids were mixed in chloroform at a molar ratio of DOPC:DOPS = 8:2. The solvent was evaporated under a nitrogen stream and further dried under vacuum for 2 hours. The resulting lipid film was resuspended in buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl₂) to form multilamellar vesicles. These were subsequently tip-sonicated for 10 minutes to produce small unilamellar vesicles (SUVs). To form supported lipid bilayers (SLBs), 5 µL of the SUV suspension (0.1 mg mL⁻¹ total lipid concentration) was deposited onto freshly cleaved mica. Following vesicle fusion, excess lipids were removed by rinsing with deionized water and then buffer. Annexin V was introduced into the imaging buffer at varying concentrations (100-200nM). All high-speed AFM measurements were performed using the NanoRacer HS-AFM (Bruker) operating in amplitude modulation mode. Imaging was conducted in liquid at ambient temperature within an acoustic isolation enclosure on an active anti-vibration table. Short cantilevers (USC-F1.2-k0.15, NanoWorld) were used, with nominal properties: Spring constant: ~0.15 N·m⁻¹; Resonance frequency: ~0.6 MHz; Quality factor: ~2 Data Access and Tools The dataset includes raw HS-AFM image sequences along with associated metadata for each acquisition. Recommended tools for accessing and analysing the data: NanoLocz PlayNano Note! Use the “Height” channel (feedback signal) rather than the “Height Measured” channel (capacitive measurement). Notes The dataset focuses on time-resolved membrane-protein assembly dynamics. Metadata fields include scan size, pixel resolution, scan speed, line rate, and imaging channel. Image files are organised in 1 zip per image stack, with corresponding entries in the metadata table: Folder Name Frames Use channel Speed (FPS) Scan Size (nm) y pixels x pixels Pixel Per nm Line Speed (Hz) Notes imaging-13.08.28.653 7 Height-ReTrace 0.78 600 256 256 0.43 200 diffusing annexin imaging-13.26.46.569 81 Height -Bi-Directional 1.56 100 256 256 2.56 200 diffusing annexin imaging-13.30.11.268 8 Height -Bi-Directional 1.56 200 256 256 1.28 200 diffusing annexin imaging-13.37.48.575 7 Height -Bi-Directional 0.78 400 512 512 1.28 200 diffusing annexin imaging-13.46.35.510 59 Height -Bi-Directional 0.78 350 512 512 1.46 200 lattice appears imaging-13.47.52.797 9 Height -Bi-Directional 0.78 200 512 512 2.56 200 lattice assembly imaging-13.48.05.546 41 Height -Bi-Directional 1.56 200 256 256 1.28 200 lattice assembly imaging-13.48.42.653 36 Height-ReTrace 0.78 200 256 256 1.28 200 lattice assembly imaging-13.49.30.677 17 Height -Bi-Directional 1.56 200 256 256 1.28 200 lattice assembly imaging-13.49.42.515 100 Height -Bi-Directional 2.73 200 256 256 1.28 350 lattice assembly imaging-13.51.38.236 75 Height -Bi-Directional 12.50 200 128 128 0.64 800 lattice assembly imaging-13.51.54.501 56 Height -Bi-Directional 15.63 200 128 128 0.64 1000 lattice assembly imaging-13.52.13.441 81 Height -Bi-Directional 11.72 200 128 128 0.64 750 lattice assembly imaging-13.52.33.276 73 Height -Bi-Directional 12.50 200 128 128 0.64 800 lattice assembly imaging-13.53.38.041 437 Height -Bi-Directional 23.44 100 64 128 1.28 750 lattice assembly imaging-13.54.14.288 436 Height -Bi-Directional 31.25 100 64 128 1.28 1000 lattice assembly imaging-13.55.22.617 490 Height -Bi-Directional 31.25 85 64 128 1.51 1000 lattice assembly imaging-13.55.40.346 762 Height -Bi-Directional 31.25 85 64 128 1.51 1000 lattice assembly imaging-14.01.11.254 15 Height -Bi-Directional 0.78 170 256 256 1.51 100 lattice assembly imaging-14.04.11.121 276 Height -Bi-Directional 23.44 120 64 128 1.07 750 lattice assembly imaging-14.14.05.504 14 Height-ReTrace 0.75 70 400 400 5.71 300 lattice imaging-14.14.26.048 5 Height-ReTrace 1.17 70 256 256 3.66 300 lattice imaging-14.14.31.069 145 Height -Bi-Directional 2.34 70 256 256 3.66 300 lattice imaging-14.15.34.569 184 Height -Bi-Directional 3.91 70 256 256 3.66 500 lattice imaging-14.16.23.572 57 Height -Bi-Directional 3.91 50 256 256 5.12 500 lattice imaging-14.16.54.040 141 Height -Bi-Directional 7.81 50 128 256 5.12 500 lattice imaging-14.20.38.415 350 Height -Bi-Directional 11.72 40 128 256 6.40 750 lattice imaging-14.21.25.854 723 Height -Bi-Directional 31.25 30 64 128 4.27 1000 lattice imaging-14.23.39.213 201 Height -Bi-Directional 54.69 20 64 128 6.40 1750 lattice imaging-14.23.44.983 2437 Height -Bi-Directional 54.69 20 64 128 6.40 1750 lattice imaging-14.25.58.071 232 Height -Bi-Directional 1.17 85 256 256 3.01 150 lattice imaging-14.29.34.756 96 Height -Bi-Directional 0.59 200 512 512 2.56 150 lattice imaging-14.37.19.845 3 Height-ReTrace 0.38 70 400 400 5.71 150 lattice imaging-14.37.28.689 5 Height-ReTrace 1.00 70 400 400 5.71 400 lattice imaging-14.37.34.764 17 Height-ReTrace 0.50 70 400 400 5.71 200 lattice imaging-14.39.16.156 6 Height-ReTrace 1.56 60 256 256 4.27 400 lattice imaging-14.39.20.305 96 Height -Bi-Directional 3.13 60 256 256 4.27 400 lattice