Cryo-electron tomography combined with image processing by sub-tomogram averaging is unique in its power to resolve the structures of proteins and macromolecular complexes in situ. Limitations of the method, including the low signal to noise ratio within individual images from cryo-tomographic datasets and difficulties in determining the defocus at which the data was collected, mean that to date the very best structures obtained by sub-tomogram averaging are limited to a resolution of approximately 15 A. Here, by optimizing data collection and defocus determination steps, we have determined the structure of assembled Mason Pfizer monkey virus Gag protein using sub-tomogram averaging to a resolution of 8.5 angstrom. At this resolution alpha-helices can be directly and clearly visualized. These data demonstrate for the first time that high-resolution structural information can be obtained from cryo-electron tomograms using sub-tomogram averaging. Sub-tomogram averaging has the potential to allow detailed studies of unsolved and biologically relevant structures under biologically relevant conditions. (C) 2013 Elsevier Inc. All rights reserved.