National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
1R01AI113321
英国
Wellcome Trust
102463/Z/13/Z
英国
Wellcome Trust
099786/Z/12/Z
英国
引用
ジャーナル: EMBO J / 年: 2022 タイトル: Helical ordering of envelope-associated proteins and glycoproteins in respiratory syncytial virus. 著者: Michaela J Conley / Judith M Short / Andrew M Burns / James Streetley / Joshua Hutchings / Saskia E Bakker / B Joanne Power / Hussain Jaffery / Joanne Haney / Giulia Zanetti / Pablo R Murcia ...著者: Michaela J Conley / Judith M Short / Andrew M Burns / James Streetley / Joshua Hutchings / Saskia E Bakker / B Joanne Power / Hussain Jaffery / Joanne Haney / Giulia Zanetti / Pablo R Murcia / Murray Stewart / Rachel Fearns / Swetha Vijayakrishnan / David Bhella / 要旨: Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image ...Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.
超分子 #1: Matrix layer of respiratory syncytial virus
超分子
名称: Matrix layer of respiratory syncytial virus / タイプ: complex / ID: 1 / 親要素: 0 詳細: Sub tomogram averaging was performed focusing on the envelope of filamentous virions propagated directly on the cryo-EM grid.
フィルム・検出器のモデル: GATAN K2 QUANTUM (4k x 4k) 検出モード: COUNTING / 撮影したグリッド数: 1 / 平均露光時間: 1.0 sec. / 平均電子線量: 2.0 e/Å2 詳細: Data collected at the UK electron bioimaging centre at Diamond Light Source (eBIC) on a Thermo-Fisher Scientific Titan Krios microscope equipped with a Gatan BioQuantum K2 energy filtered ...詳細: Data collected at the UK electron bioimaging centre at Diamond Light Source (eBIC) on a Thermo-Fisher Scientific Titan Krios microscope equipped with a Gatan BioQuantum K2 energy filtered direct detection camera. Dose-symmetric tilt-series collection was performed using SerialEM. Energy-filtered images were collected with a slit-width of 20eV and an applied defocus of between -2 and -4.5 microns. One second exposures were recorded with an electron exposure of 2 electrons/A2, partitioned over five movie frames. A total of 41 images were recorded per tilt-series at 3 degree intervals from -60 to +60, thus a total exposure of 82 electrons/A2 was applied per tilt-series. Tilt-series were recorded at a nominal column magnification of 81k corresponding to a calibrated pixel size of 1.79 A at the specimen scale.
Individual movies were corrected for drift and beam induced motion using MotionCor2. Motion corrected images were then compiled into an angle ordered stack file using a perl script to extract the correct files and tilt angles from the SerialEM metadata files (mdoc), creating the final tilt-series using the IMOD command new-stack. Tilt-series alignment and reconstruction by weighted back projection was then accomplished using the IMOD package
最終 再構成
想定した対称性 - 点群: C1 (非対称) / 解像度のタイプ: BY AUTHOR / 解像度: 16.0 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: Dynamo 詳細: Half-maps generated using Dynamo and resolution assessment, low-pass filtering performed using Relion. 使用したサブトモグラム数: 30082
抽出
トモグラム数: 10 / 使用した粒子像数: 30088 / ソフトウェア - 名称: Dynamo 詳細: Selected using crop on rings along path model in Dynamo