Pushing Single-Particle Cryo-EM To The Theoretical Size and Resolution Limits At 200 keV Mengyu Wu1†, Mark A. Herzik, Jr.2†, Gabriel C. Lander1*

1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 2. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA † These authors contributed equally to this work * Corresponding author: glander@scripps.edu Recent technical advances in cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) have enabled the direct visualization of biological macromolecules in near-native states at increasingly higher resolutions, propelling the technique towards the forefront of structural biology. Notably, cryo-EM enables 3D structure determination of specimens in a vitrified state without the requirement of crystallization [1], allowing for visualization of complexes previously deemed intractable for structural studies due to size, conformational heterogeneity, and/or compositional variability [2-4]. Indeed, determining ~3 Å reconstructions of stable specimens by SPA has become increasingly routine, garnering much interest in the resolution limit of this technique. Several recent studies have demonstrated that resolutions of ~2 Å or better can be achieved using a 300 keV transmission electron microscope (TEM) such as the Titan Krios equipped with a direct electron detector (DED) [5-7]. Comparatively, the capabilities of TEMs operating at 200 keV for resolving biological specimens have not been extensively examined. To complement existing work, we investigated the resolution limit of a 200 keV TEM (Talos Arctica) equipped with a K2 Summit DED using mouse heavy-chain apoferritin as a test specimen. Through systematic testing of various data collection and processing parameters, including refinement of pixel size and the spherical aberration coefficient (Cs) values, we demonstrate that 1.8 Å resolution can be achieved using this instrumentation. The results presented in this study prove that resolutions comparable to those obtained from 300 keV TEMs can be attained using 200 keV systems, and will support future technical studies in the field. Despite significant advances in resolution, cryo-EM remains limited by specimen size due to the low signal-to-noise ratio (SNR) of cryo-EM images. Indeed, although SPA reconstructions of molecules as small as 38 kilodaltons (kDa) have been theorized to be achievable [8], this feat has yet to be realized. Though Volta Phase Plate technology has enabled visualization of specimens in this size range [4], this instrumentation is not yet fully automated and can present technical challenges. Due to the limited success in imaging smaller macromolecules by cryo-EM, the technique has primarily been used to visualize large complexes; to date, only three macromolecular complexes smaller than 100 kDa have been resolved to high resolution (i.e. better than 4 Å) using SPA [4,9-10]. We expand upon our previous work [11] and demonstrate that conventional defocus-based cryo-EM methodologies can be used to determine high-resolution structures of specimens amassing <100 kDa using a base model (i.e. excluding imaging accessories such as a phase plate or energy filter) Talos Arctica TEM equipped with a K2 Summit. We determined the structure of 82 kDa alcohol dehydrogenase to ~2.7 Å resolution, proving that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions using SPA (Figure 1A). We also determined ~2.8 Å and ~3.2 Å structures of two conformational species of

 

methemoglobin and demonstrate that distinct functional states can be identified within a dataset for proteins as small as 64 kDa, underscoring the utility of SPA in examining the conformational dynamics of similarly small biological systems (Figure 1B). Finally, we provide the first sub-nanometer structure of a sub-50 kDa macromolecular complex – the 43 kDa catalytic domain of protein kinase A (Figure 1C). These results define a new frontier for target sizes that can feasibly be resolved using conventional cryo-EM without the need for a phase plate or use of significant underfocus (e.g. >2000 nm). Taken together, these findings broaden the potential of cryo-EM as a powerful tool for a variety of structure-based studies, particularly in drug discovery. Our work further propels an important trajectory for cryo-EM SPA and indicates that high-resolution structure determination of complexes approaching, or even exceeding, the theoretical size limit of cryo-EM will likely be realized in the near future [12]. References: [1] Nogales, E. and Scheres, S. H. W., Mol Cell 58 (2015), p. 677–689. [2] Lander, G. C. et al., Nature 482 (2012), p. 186–191. [3] Yan, C. et al., Science 353 (2016), p. 904–911. [4] Khoshouei, M. et al., Nat Commun 8 (2017), p. 16099. [5] Bartesaghi, A. et al., Science 348 (2015), p. 1147–1151. [6] Bartesaghi, A. et al., Structure 26 (2018), p. 848–856.e3. [7] Tan, Y. Z. et al., Nat Commun 9 (2018), p. 3628. [8] Henderson, R., Q. Rev. Biophys. 28 (1995), p. 171–193. [9] Merk, A. et al,. Cell 165 (2016), p. 1698–1707. [10] Fan, X. et al., Biorxiv preprint (2018), https://doi.org/10.1101/457861. [11] Herzik, M. A., Wu, M. and Lander, G. C., Nat Methods 14 (2017), p. 1075–1078. [12] The authors acknowledge J.C. Ducom at The Scripps Research Institute (TSRI) High Performance

Computing facility for computational support, and B. Anderson at the TSRI electron microscopy facility for microscope support. We thank P. Aoto of the S. Taylor laboratory (University of California, San Diego) for kindly providing iPKAc for this study.

Figure 1. High-resolution structure determination of sub-100 kDa complexes using defocus-based cryo-EM SPA methodologies. A. ~2.7 Å resolution cryo-EM reconstruction of 82 kDa horse liver alcohol dehydrogenase, colored by subunit. The segmented NADH EM density is shown in yellow. Zoomed-in views of the EM density for the active-site zinc (inset, upper left), structural zinc site (inset, upper right), and active site (inset, bottom). B. ~2.8 Å resolution cryo-EM reconstruction of 64 kDa human methemoglobin, colored by subunit with the segmented EM density for the heme cofactors colored gray. C. ~6 Å cryo-EM reconstruction of 43 kDa catalytic domain of protein kinase A, shown as a transparent gray surface with the fitted atomic model (PDB ID: 1ATP) shown as a blue cartoon.