Journal: Mol Cell / Year: 2015 Title: Cryo-EM structure of influenza virus RNA polymerase complex at 4.3 Å resolution. Authors: Shenghai Chang / Dapeng Sun / Huanhuan Liang / Jia Wang / Jun Li / Lu Guo / Xiangli Wang / Chengcheng Guan / Bhargavi M Boruah / Lingmin Yuan / Feng Feng / Mingrui Yang / Lulan Wang / Yao ...Authors: Shenghai Chang / Dapeng Sun / Huanhuan Liang / Jia Wang / Jun Li / Lu Guo / Xiangli Wang / Chengcheng Guan / Bhargavi M Boruah / Lingmin Yuan / Feng Feng / Mingrui Yang / Lulan Wang / Yao Wang / Justyna Wojdyla / Lanjuan Li / Jiawei Wang / Meitian Wang / Genhong Cheng / Hong-Wei Wang / Yingfang Liu / Abstract: Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the ...Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the underlying mechanism of the RdRP complex is still unclear. Here we report the biochemical characterization of influenza RdRP subcomplex comprising PA, PB1, and N terminus of PB2, which exist as dimer in solution and can assemble into a tetramer state, regulated by vRNA promoter. Using single-particle cryo-electron microscopy, we have reconstructed the RdRP tetramer complex at 4.3 Å, highlighting the assembly and interfaces between monomers within the tetrameric structure. The individual RdRP subcomplex contains all the characterized motifs and appears as a cage-like structure. High-throughput mutagenesis profiling revealed that residues involved in the oligomer state formation are critical for viral life cycle. Our results lay a solid base for understanding the mechanism of replication of influenza and other negative-stranded RNA viruses.
History
Deposition
Nov 28, 2014
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Header (metadata) release
Dec 31, 2014
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Map release
Feb 4, 2015
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Update
Jun 3, 2015
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Current status
Jun 3, 2015
Processing site: PDBj / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
pH: 7.8 / Details: 25 mM HEPES 200 mM NaCl and 1 mM EDTA
Grid
Details: Quantifoil Cu R2/1
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV Method: The grids were blotted for 6.5 s with 100% humidity and flash frozen in liquid ethane cooled at liquid nitrogen temperature using an FEI Vitrobot.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Date
Oct 2, 2014
Image recording
Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 50 e/Å2 Details: Each image was fractionated into 32 sub-frames, with 0.25 s exposure time per frame. All dose-fractionated cryo-EM images were recorded using a semi-automated low-dose acquisition program UCSF-Image4
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 22500
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