Finding trimeric HIV-1 envelope glycoproteins in random noise

Abstract

In two recent papers, Mao et al. (1, 2) present the trimeric structure of the HIV-1 gp160 trimer at 11- and 6-A resolutions, respectively. The authors repeatedly emphasize their “reference-free,” “gold standard” methodology, but in neither of their papers do they state how the particles were selected from the electron micrographs. They do state (figure S3 in ref. 1): “Each row shows a sequence of average images from the same projection class, progressing from the initial average to the converged average,” and “refinements started without an external or prior reference images and did not assume any symmetry.” These projection classes thus must have existed prior to the refinements, as the result of an explicit search for specific molecular views, namely projection images generated from an earlier 3D structure. Because the reference bias is introduced at this earlier stage, it becomes irrelevant that the projection classes can be averaged without using further references or symmetry assumptions. In contrast to local-variance particle detection (3), correlation function picking looks for specific views in specific orientations and may introduce reference bias (4), as do all forms of reference-based alignment (4, 5). My best guess is that an earlier structure (EMDataBank EMD-5019) was used to pick particles in ref. 1.