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-Structure paper
タイトル | Overcoming resolution attenuation during tilted cryo-EM data collection. |
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ジャーナル・号・ページ | Nat Commun, Vol. 15, Issue 1, Page 389, Year 2024 |
掲載日 | 2024年1月9日 |
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 / |
PubMed 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. |
リンク | Nat Commun / PubMed:38195598 / PubMed Central |
手法 | EM (単粒子) |
解像度 | 1.92 - 3.2 Å |
構造データ | EMDB-36766: Untilted (0 Degree Tilted) Single-Particle CryoEM Reconstruction of AAV2 Viral Capsid EMDB-36767: 10 Degree Tilted Single-Particle CryoEM Reconstruction of AAV2 Viral Capsid EMDB-36768: 20 Degree Tilted Single-Particle CryoEM Reconstruction of AAV2 Viral Capsid EMDB-36769: 30 Degree Tilted Single-Particle CryoEM Reconstruction of AAV2 Viral Capsid EMDB-36770: 40 Degree Tilted Single-Particle CryoEM Reconstruction of AAV2 Viral Capsid EMDB-36771: 50 Degree Tilted Single-Particle CryoEM Reconstruction of AAV2 Viral Capsid EMDB-36772: 60 Degree Tilted Single-Particle CryoEM Reconstruction of AAV2 Viral Capsid EMDB-36807: Untilted (0 Degree Tilted) Single-Particle CryoEM Reconstruction of Apoferritin EMDB-36809: 10 Degree Tilted Single-Particle CryoEM Reconstruction of Apoferritin EMDB-36810: 20 Degree Tilted Single-Particle CryoEM Reconstruction of Apoferritin EMDB-36811: 30 Degree Tilted Single-Particle CryoEM Reconstruction of Apoferritin EMDB-36812: 40 Degree Tilted Single-Particle CryoEM Reconstruction of Apoferritin EMDB-36813: 60 Degree Tilted Single-Particle CryoEM Reconstruction of Apoferritin EMDB-36814: 50 Degree Tilted Single-Particle CryoEM Reconstruction of Apoferritin EMDB-36816: Untilted (0 Degree Tilted) Single-Particle CryoEM Reconstruction of DPS EMDB-36817: 10 Degree Tilted Single-Particle CryoEM Reconstruction of DPS EMDB-36818: 20 Degree Tilted Single-Particle CryoEM Reconstruction of DPS EMDB-36819: 30 Degree Tilted Single-Particle CryoEM Reconstruction of DPS EMDB-36820: 40 Degree Tilted Single-Particle CryoEM Reconstruction of DPS EMDB-36821: 50 Degree Tilted Single-Particle CryoEM Reconstruction of DPS EMDB-36822: 60 Degree Tilted Single-Particle CryoEM Reconstruction of DPS EMDB-41230: Untilted (0 Degree Tilted) Single-Particle CryoEM Reconstruction of Apoferritin EMDB-41231: 30 Degree Tilted Single-Particle CryoEM Reconstruction of Apoferritin EMDB-41695: 60 Degree Tilted Single-Particle CryoEM Reconstruction of RNA polymerase |
化合物 | ChemComp-ZN: ChemComp-MG: ChemComp-S9F: |
由来 |
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キーワード | TRANSCRIPTION / complex / unnatural base pairs / synthetic biology |