The study investigates the structural conversion of the C-terminal (CT) domain of major ampullate Spidroin 1 (MaSp1) during the assembly of spider silk fibers. The CT domain, crucial for fiber formation, undergoes an α-helix to β-sheet transition upon fiber formation, with helix N°4 being the most likely initiating segment. This transition is supported by circular dichroism (CD) spectroscopy, which shows a shift from α-helical to β-sheet content. The disulfide bond in helix N°4 aligns the chains in a parallel manner, potentially acting as a nucleation point for the structural conversion. X-ray diffraction patterns of CT fibers reveal a distinct structure compared to natural spider silk, indicating a mixture of β-sheet and nanocrystalline components. Solid-state nuclear magnetic resonance (ssNMR) studies confirm the presence of β-sheets and α-helices in CT fibers, with intermolecular backbone contacts observed. Computational analysis using ZipperDB and TANGO algorithms suggests that helix N°4 has a high propensity for forming β-sheets, supported by the synthesis and analysis of a synthetic peptide corresponding to this region. The peptide exhibits pH-dependent α-to-β structural conversion and assembles into nanofibrils, further validating the role of helix N°4 in the structural transition. These findings provide insights into the structural basis of fiber formation in spider silk and highlight the importance of helix N°4 in the assembly process.The study investigates the structural conversion of the C-terminal (CT) domain of major ampullate Spidroin 1 (MaSp1) during the assembly of spider silk fibers. The CT domain, crucial for fiber formation, undergoes an α-helix to β-sheet transition upon fiber formation, with helix N°4 being the most likely initiating segment. This transition is supported by circular dichroism (CD) spectroscopy, which shows a shift from α-helical to β-sheet content. The disulfide bond in helix N°4 aligns the chains in a parallel manner, potentially acting as a nucleation point for the structural conversion. X-ray diffraction patterns of CT fibers reveal a distinct structure compared to natural spider silk, indicating a mixture of β-sheet and nanocrystalline components. Solid-state nuclear magnetic resonance (ssNMR) studies confirm the presence of β-sheets and α-helices in CT fibers, with intermolecular backbone contacts observed. Computational analysis using ZipperDB and TANGO algorithms suggests that helix N°4 has a high propensity for forming β-sheets, supported by the synthesis and analysis of a synthetic peptide corresponding to this region. The peptide exhibits pH-dependent α-to-β structural conversion and assembles into nanofibrils, further validating the role of helix N°4 in the structural transition. These findings provide insights into the structural basis of fiber formation in spider silk and highlight the importance of helix N°4 in the assembly process.