Human adenoviruses (HAdVs) frequently cause infections of the respiratory tract, gastrointestinal tract and eyes [1]. While CAR (coxsackie and adenovirus receptor) acts as receptor for most adenoviruses, entry of species C into polarized epithelial cells which lack CAR remained unknown. Recently it was discovered that HAdV-C5 blankets itself with lactoferrin (LF), an iron-binding innate immune protein, and exploits NCL, a natural receptor of lactoferrin, to infect host cells [1]. AdVs have been successfully utilised in vaccines against SARS-CoV2 and are of interests in the field of gene therapy. HAdV-C5 must bind to LF first via its major capsid protein, Hexon (AdV5H) in order to exploit NCL for entry into epithelial cells. Binding of AdV5 to NCL via LF is a 2-step process. LF has to bind to the virus first before the cellular receptor can be engaged. LF bound to NCL is unable to enhance viral entry via NCL. We aim to understand the mechanism of NCL mediated entry of HAdV-C5 into host cells via LF by determining the structure of HAdV-C5 Hexon: Lactoferrin: Nulceolin (AdV5H: LF: NCL) tripartite complex. Towards this aim, we first set out to prepare the AdV5H: LF, and HAdV-C5: LF complex and determine the cryo-EM structure. We employ mass spectrometry, microscale thermophoresis, and electron microscopy to characterise the structure of AdV5H: LF, and HAdV-C5: LF complex.