National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
U54 AI170855
米国
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
U01 AI136680
米国
National Institutes of Health/Office of the Director
GM148476
米国
National Institutes of Health/Office of the Director
GM082946
米国
National Science Foundation (NSF, United States)
MCB 2048095
米国
Other private
Hearst Foundations Developmental Chair
Other private
Margaret T. Morris Foundation
Other government
National University of Singapore (A-0008405-00-00, A-0008405-01-00)
Ministry of Education (MoE, Singapore)
A-8000037-00-00
シンガポール
National Research Foundation (NRF, Singapore)
A-8001346-00-00
シンガポール
引用
ジャーナル: Nat Commun / 年: 2024 タイトル: Overcoming resolution attenuation during tilted cryo-EM data collection. 著者: 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 ...著者: 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 / Dmitry Lyumkis / 要旨: Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding ...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.