Negative stain TEM imaging of native spider silk protein superstructures

Native Latrodectus hesperus (black widow) major ampullate spider silk proteins were imaged using negative stain transmission electron microscopy (NS-TEM) by isolating the silk protein hydrogel directly from the organism and solubilizing in urea. Heterogeneous micelle-like structures averaging 300 nm, similar to those imaged previously with CryoEM, were observed when stained with ammonium molybdate. A second smaller population averaging 50 nm was observed as well as large fibrils, highlighting the heterogeneous nature of the silk gland. The population of smaller silk protein micelles was enhanced at higher urea concentrations (5-8 M). This was further supported by dynamic light scattering (DLS), where two populations were observed at low urea concentrations while one small population dominated at high urea concentrations. The approach presented here provides a cost-effective route to imaging silk protein superstructures with conventional NS-TEM methods and may be applicable to other soft nanoparticle systems.