Single particle cryo-electron microscopy (cryo-EM) allows not only for near-atomic structure determination of large protein complexes preserved in vitreous ice, but recently it was shown to provide a solution for the structure determination of inherently heterogeneous protein molecules via segregation of conformational variation into a set of 3D classes. In general workflow of cryo-EM image processing, 3D classification is employed for the selection of the dataset that belong in the best 3D class, followed by final 3D reconstruction through iterative refinements. However, image processing of structurally heterogeneous protein complexes requires a series of 3D classification both pre- and post-refinement in a hierarchical manner, as well as the detection of local disorder that are resolved by focused refinement. Here we describe the application of such workflow that effectively resolved structural heterogeneity of RNA polymerase (RNAP) from Thermococcus kodakarensis. Cryo-EM structures of RNAP in its apo form and in complex with transcription factor E (TFE), both at approximately 4Å resolution, demonstrates concerted disposition of subunits that corresponds to stalk and clamp region upon binding of TFE. In addition, focused refinement revealed secondary structure arrangements at the binding interface between RNAP and TFE. This local movement is deemed to provide accessibility to DNA at cleft region hence allowing for the formation of preinitiation complex.