Overcoming resolution attenuation during tilted cryo-EM data collection

Sriram Aiyer, Philip R. Baldwin, Shi Min Tan, Zelin Shan, Juntaek Oh, Atousa Mehrani, Marianne E. Bowman, Gordon Louie, Dario Oliveira Passos, Selena Đorđević-Marquardt, Mario Mietzsch, Joshua A. Hull, Shuichi Hoshika, Benjamin A. Barad, Danielle A. Grotjahn, Robert McKenna, Mavis Agbandje-McKenna, Steven A. Benner, Joseph A. P. Noel, Dong Wang, Yong Zi Tan () and Dmitry Lyumkis ()
Additional contact information
Sriram Aiyer: Laboratory of Genetics, The Salk Institute for Biological Studies
Philip R. Baldwin: Laboratory of Genetics, The Salk Institute for Biological Studies
Shi Min Tan: National University of Singapore
Zelin Shan: Laboratory of Genetics, The Salk Institute for Biological Studies
Juntaek Oh: University of California, San Diego
Atousa Mehrani: Laboratory of Genetics, The Salk Institute for Biological Studies
Marianne E. Bowman: Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies
Gordon Louie: Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies
Dario Oliveira Passos: Laboratory of Genetics, The Salk Institute for Biological Studies
Selena Đorđević-Marquardt: Laboratory of Genetics, The Salk Institute for Biological Studies
Mario Mietzsch: University of Florida
Joshua A. Hull: University of Florida
Shuichi Hoshika: Foundation for Applied Molecular Evolution, 13709 Progress Blvd Box 7
Benjamin A. Barad: The Scripps Research Institute
Danielle A. Grotjahn: The Scripps Research Institute
Robert McKenna: University of Florida
Mavis Agbandje-McKenna: University of Florida
Steven A. Benner: Foundation for Applied Molecular Evolution, 13709 Progress Blvd Box 7
Joseph A. P. Noel: Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies
Dong Wang: University of California, San Diego
Yong Zi Tan: National University of Singapore
Dmitry Lyumkis: Laboratory of Genetics, The Salk Institute for Biological Studies

Nature Communications, 2024, vol. 15, issue 1, 1-19

Abstract: Abstract Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting “preferred orientations” on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

Date: 2024
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DOI: 10.1038/s41467-023-44555-7

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