Accurately determining a cryoEM particle's alignment parameters is crucial to high resolution single particle 3-D reconstruction. We developed Multi-Path Simulated Annealing, a Monte-Carlo type of optimization algorithm, for globally aligning the center and orientation of a particle simultaneously. A consistency criterion was developed to ensure the alignment parameters are correct and to remove some bad particles from a large pool of images of icosahedral particles. Without using any a priori model, this procedure is able to reconstruct a structure from a random initial model. Combining the procedure above with a new empirical double threshold particle selection method, we are able to pick tens of best quality particles to reconstruct a subnanometer resolution map from scratch. Using the best 62 particles of rice dwarf virus, the reconstruction reached 9.6 angstrom resolution at which four helices of the P3A subunit of RDV are resolved. Furthermore, with the 284 best particles, the reconstruction is improved to 7.9 angstrom resolution, and 21 of 22 helices and six of seven P sheets are resolved. (c) 2007 Elsevier Inc. All rights reserved.