(a) The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex traverses the nuclear envelope to transmit forces between the cytoskeleton and nuclear components. The canonical model of the LINC complex is a linear structure formed of SUN and Nesprin proteins, which interact via a 3:3 complex between their SUN and KASH domains within the peri-nuclear space, and cross the inner and outer nuclear membranes (INM and ONM), respectively. (b) Sequence alignment of the KASH domains of human Nesprins 1–4 and KASH5. In this study, KASH1, KASH4, and KASH5 refer to the C-terminal KASH domains of Nesprin-1, Nesprin-4, and KASH5, respectively, which are highlighted (black outline), and key amino acids within KASH4 and KASH5 are indicated. (c) Crystal structures of human SUN1-KASH4 (top), SUN1-KASH5 (middle), and SUN1-KASH1 (bottom). The SUN1 molecular surface is displayed with SUN1 KASH-lids highlighted in blue as cartoons, and KASH sequences are represented as sticks (yellow, purple, and red, respectively). All structures are 6:6 complexes in which KASH proteins lie at the midline head-to-head interface between SUN1 trimers. (d) SEC-MALS analysis showing differential refractive index (dRI) profiles with fitted molecular weights (Mw) plotted as diamonds across elution peaks. SUN1-KASH4, SUN1-KASH5, and SUN1-KASH1 form 6:6 complexes in solution, with experimental molecular weights of 150, 154, and 156 kDa, respectively (theoretical 6:6 – 155, 155, and 157 kDa). Representative of more than three replicates using different protein preparations. Full elution profiles are shown in Figure 1—figure supplement 2.