Microtubules (MTs) are cylindrical polymers of alpha beta-tubulin that display pseudo-helical symmetry due to the presence of a lattice seam of heterologous lateral contacts. The structural similarity between alpha- and beta-tubulin makes it difficult to computationally distinguish them in the noisy cryo-EM images, unless a marker protein for the tubulin dimer, such as kinesin motor domain, is present. We have developed a new data processing protocol that can accurately determine alpha beta-tubulin register and seam location for MT segments. Our strategy can deal with difficult situations, where the marker protein is relatively small or the decoration of marker protein is sparse. Using this new seam-search protocol, combined with movie processing for data from a direct electron detection camera, we were able to determine the cryo-EM structures of MT at 3.5 angstrom resolution in different functional states. The successful distinction of alpha- and beta-tubulin allowed us to visualize the nucleotide state at the E-site and the configuration of lateral contacts at the seam. (C) 2015 Elsevier Inc. All rights reserved.