1. Markey Center for Structural Biology, Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA
2. National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
4. These authors contributed equally to this work.

Correspondence to: Wen Jiang^1,4Wah Chiu² Correspondence and requests for materials should be addressed to W.C. (Email: wah@bcm.edu) or W.J. (Email: jiang12@purdue.edu).

A half-century after the determination of the first three-dimensional crystal structure of a protein¹, more than 40,000 structures ranging from single polypeptides to large assemblies have been reported². The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-Å resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (15) particle, by single-particle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity^{3, 4}. However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond 15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.

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