Cryo-electron microscopy has undergone significant advances in hardware development that push limits of 3-D structure determination of large macromolecular complexes of proteins and nucleic acids. New generation of electron microscopes (such as FEI Titan Krios) with parallel sample illumination, stable electro-magnetic lenses and new specimen compustage provided a platform for obtaining data extending to near-atomic resolution. Additionally, advent of direct detectors with 4-times better sensitivity and capability to record several images per second revolutionized the field of cryo-electron microscopy of biological specimen.
We used this new instrumentation to image the type I restriction-modification enzyme EcoR124I that is at the edge of standard cryoEM procedures and reconstructions: it is only ~500 kDa large, has low symmetry and most likely is flexible in its open conformation [1]. The data obtained from Titan Krios with a direct detector provided data that can be processed and lead to high-resolution 3D reconstruction. However, we had to develop a new approch for processing of the aquired data to extract the individual EcoR124I particles from imaged micrographs. We used wavelet filter (i.e. dyadic and stationary wavelet transform with different treasholds) to enhance the contrast of the particles in the micrograph that facilitated identification of their positions and automated picking from thousands of collected micrographs. Next, we obtained the initial low-resolution model of the EcoR124I structure from cryo-electron tomography, a technique that allows 3-D reconstruction without any initial assumption about the 3-D structure. The average from ~300 subtomogram particles was used to automatically locate particles in the filtered micrographs, using the algorithm implemented in the image processing package Relion [2]. Our next efforts are focused on refining the 3D structure to subnanometer resolution using the original dataset obtained from Titan Krios.