Determination of the structure of very large protein assemblies, such as the nuclear pore complex (NPC), remains quite challenging because current methods used to determine their structures in situ lack the resolution needed for detailed molecular assignment. To address this, Ellenberg and colleagues have developed a methodological approach that combines super-resolution fluorescence microscopy with single-particle averaging. They used their method to examine the organization of the NPC scaffold primarily composed of multiple copies of the Nup107–160 subcomplex. By combining thousands of super-resolution images of single nuclear pores to generate an average image, it was possible to map the average position of a given fluorescent label in the NPC with a precision of 0.1 nm and an accuracy of 0.3 nm. The Nup107–160 subcomplex is believed to be structurally homologous to that of the yeast Nup84 subcomplex, which crystallographic, electron microscopic and biochemical work have revealed to be Y-shaped. The authors first attempted to identify members of the complex by using fluorescent-labeled antibodies. The data suggested that the stalk (Nup133 and Nup107) and the Nup160 arm are located at the periphery of the nuclear pore, with the second, Nup85–Seh1 arm reaching into the center. Further examination using Nup-GFP fusions and dye-coupled anti-GFP nanobodies indicated that the long stalk of the Y was oriented perpendicularly to the direction of transport through the NPC. When examined in conjunction with the cryo-EM structure of the human NPC, the data support a head-to-tail arrangement of the Nup107–160 complex in the cytoplasmic and nucleoplasmic rings of the pore, with 8 or 16 copies of the complex in each ring, and do not support previously proposed fence-like or lattice models. This methodology could bridge the gap between atomic resolution techniques and label-free in situ methods. (Sciencehttp://dx.doi.org/10.1126/science.1240672,publishedonline11July2013)