It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of similar to 10 angstrom, but maps with resolutions of 5 angstrom or better are still celebrated events. The electron microscope has a resolving power to better than 2 angstrom, and thus should not be a limiting factor; instead the practical limitations in resolution most likely arise from a combination of specimen preparation methods, data collection parameters, and data analysis procedures. With the aid of a highly automated system for acquiring images, coupled to a relational database to keep track of all processing parameters, we have taken a systematic approach to optimizing parameters affecting the resolution of single particle reconstructions. Using GroEL as a test-bed, we performed a series of 3D reconstructions where we systematically varied the number of particles used in computing the map, the accelerating voltage of the microscope, and the electron dose used to acquire the images. We also investigated methods for excluding unacceptable or "bad" particles from contributing to the final 3D map. Using relatively standard instrumentation (Tecnai F20, 4K x 4K CCD, side entry cold stage) and a completely automated approach, these approaches resulted in a map with a nominal resolution of 5.4 angstrom (FSC0.5) in which secondary structure is clearly discernable and the handedness of some of the alpha-helices in the GroEL structure can be determined. (C) 2008 Elsevier Inc. All rights reserved.