EMAN is a general-purpose scientific image-processing suite developed primarily for the TEM community [1]. With over ½ million lines of Python and C++ code, hundreds of image processing algorithms and a cross-platform graphical interface, it is a capable tool for quantitative greyscale image analysis in 2-D or 3-D.Over the last three years, the Cryo-EM community has begun shifting focus from improving resolution, to validation of results. EMAN2.1 represents the results of major new developments in EMAN2's single particle reconstruction and single particle tomography workflows. The new tools such as e2refine-easy, integrate "gold standard" resolution assessment into the refinement process along with a number of new optimizations, both speeding the refinement process, and eliminating the need for empirical filtration of reconstructions by users. EMAN2.1 also integrates support for tilt-pair validation and "true resolution" testing to insure self-consistency among data and final 3-D maps. This is one of the few methods which can identify incorrect maps at low resolution.Another approach often applied to increase confidence in a 3-D structure is to reprocess the same data using multiple algorithms, preferably based on different mathematical methods. EMAN2.1 includes an interface for converting data and metadata into the appropriate format for reprocessing in Relion [2] or FreAlign[3], two alternative single particle reconstruction packages. Once these packages complete their refinements, the results can be imported back into EMAN2.1 for comparison and analysis. As an alternative, EMAN2.1 can perform the opposite process as well. A refinement completed originally in one of these other packages can be converted into an EMAN2.1 project, which can then be used to re-refine the data from scratch.Another important area of improvement is single particle tomography. Rather than the traditional approach of reconstructing large numbers of 2-D images of identical particles in random orientations, in single particle tomography, tomographic data is collected for fields of particles, producing a low resolution and incomplete, but 3-D reconstruction for each individual molecule. EMAN2.1 now incorporates tools for subtomogram extraction, and a variety of different approaches for alignement and averaging of particles. This approach is a powerful alternative to single particle analysis particularly in cases where the particles are flexible or heterogeneous in solution.EMAN2.1 also incorporates several important ease-of-use improvements. At the request of our users, the EMAN2.0 strategy for storing image data and metadata has been retired in favor of flat HDF and JSON formatted files. The project manager and file browser were both rewritten for speed and 832

Single particle tomography (SPT or subtomogram averaging) offers a powerful alternative to traditional 2-D single particle reconstruction for studying conformationally or compositionally heterogeneous macromolecules. It can also provide direct observation (without labeling or staining) of complexes inside cells at nanometer resolution. The development of computational methods and tools for SPT remains an area of active research. Here we present the EMAN2.1 SPT toolbox, which offers a full SPT processing pipeline, from particle picking to post-alignment analysis of subtomogram averages, automating most steps. Different algorithm combinations can be applied at each step, providing versatility and allowing for procedural cross-testing and specimen-specific strategies. Alignment methods include all-vs-all, binary tree, iterative singlemodel refinement, multiple-model refinement, and self-symmetry alignment. An efficient angular search, Graphic Processing Unit (GPU) acceleration and both threaded and distributed parallelism are provided to speed up processing. Finally, automated simulations, per particle reconstruction of subtiltseries, and per-particle Contrast Transfer Function (CTF) correction have been implemented. Processing examples using both real and simulated data are shown for several structures.