Improving Resolution and Field of View of Electron Cryo-Microscopy With a Larger Direct Detection Device B.E. Bammes1, D.H. Chen1, L. Jin1, R. Bilhorn1 1Direct Electron, LP, San Diego, California 92128 USA Electron cryo-microscopy (cryo-EM) is a powerful tool for studying biological macromolecules with resolution ranging from nanometer to near-atomic [1]. However, the performance of the cryo-EM method still lags far behind its theoretical limit from physics of electron scattering [2]. Multiple factors reduce the overall signal-to-noise ratio (SNR) of each particle image, including radiation damage of the specimen, beam-induced specimen motion, and inefficient electron detectors [2]. The result is that, while cryo-EM is powerful, it remains tedious—often requiring significant imaging time and computational resources in order to generate an average three-dimensional (3D) high-resolution structure from millions of asymmetric units. With the goal of overcoming many of these obstacles, Direct Electron introduced the DE-12 in 2010 as the first commercial direct electron detector for cryo-EM [3]. Performance evaluations using graphitized carbon, thin carbon film, the GroEL chaperonin, and icosahedral viruses have demonstrated significantly improved SNR from the DE-12 compared to other electronic detectors [4,5]. Furthermore, the DE-12 was recently used to generate single-particle 3D reconstructions of two different icosahedral viruses—ε15 and P22—to resolutions corresponding to about 4/5 Nyquist [5]. These two structures are currently the only single-particle cryo-EM reconstructions to reach subnanometer resolution using images acquired at low magnification (20,000× or less). The ability of the DE-12 to record usable signal at and beyond 4/5 Nyquist allows users image at lower magnification, resulting in a larger field-of-view and improved imaging efficiency (Fig. 1). In addition to significantly improved SNR in each image, the DE-12 also provides “movie-mode,” allowing users to record a continuous series of frames during a single exposure. This seamless exposure fractionation opens many possibilities for new experimental methods and computational techniques in cryo-EM to further boost the usable information content of each image. Notably, “movie-mode” allows users to visualize and correct beam-induced specimen motion [6], which is one of the limiting factors to the performance of cryo-EM [2]. We have demonstrated the benefits of this strategy by using a piezoelectric control to intentionally introduce specimen drift. Images of carbon film were collected at 25 frames per second (fps) over ~2 seconds exposure time. As expected, the Fourier transform of the sum of all 48 frames showed obvious signs of specimen drift, with signal degradation in direction of the drift. To recover this lost information, each set of 4 frames were summed and oversampled by a factor of 2, and the resulting partial-sums were translationally aligned before generating the final summed image. The Fourier transform of the resulting image showed isometric Thon rings without signs of drift (Fig. 2). As the next step in advancing cryo-EM performance, Direct Electron is now introducing the DE-20 camera, which represents the largest CMOS-based direct electron detector currently produced, with 5120 × 3840 pixels (20 megapixel). This Direct Detection Device (DDD) retains the features of the DE-12, but with 1.6 times the number of pixels. The DE-20 operates with full-frame acquisition at

30 fps. A Faraday plate is included above the image sensor to detect the total electron exposure for each image. As an additional tool for diffraction and search/focus operations (especially in automated data collection), an off-axis 2k × 2k scintillator-coupled CMOS sensor is also included. References: [1] Chang, J., Liu, X., Rochat, R.H., et al., “Reconstructing virus structures from nanometer to near-atomic resolutions with cryo-electron microscopy and tomography,” Adv Exp Med Biol 726, 49-90 (2012). [2] Glaeser, R.M., Hall, R.J., “Reaching the information limit in cryo-EM of biological macromolecules: experimental aspects,” Biophys J 100, 2331-7 (2011). [3] Jin, L., Bilhorn, R., “Performance of the DDD as a direct electron detector for low dose electron microscopy,” Microsc Microanal 16, 854-5 (2010). [4] Milazzo, A.C., Cheng, A., Moeller, A., et al., “Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy,” J Struct Biol 176, 404-8 (2011). [5] Bammes, B.E., Rochat, R.H., Jakana, J., et al., “Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency,” J Struct Biol 177, 589-601 (2012). [6] Brilot, A.F., Chen, J.Z., Cheng, A., “Beam-induced motion of vitrified specimen on holey carbon film,” J Struct Biol 177, 630-7 (2012).

Fig. 1. The specimen field-of-view of several electron detectors. For any given target resolution, the DE-20 provides 1.4× and 1.7× the field-of-view as the competing 4k CMOS or 4k CCD detectors, respectively.

Fig. 2. Fourier transforms of the raw sum (left) and the sum of aligned frames (right) from a two second exposure of carbon film with significant specimen drift. Summing translationally-aligned frames results in isotropic Thon rings with no sign of drift.