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- EMDB-3291: Sub-tomogram averaging of Lassa virus glycoprotein spike from vir... -

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Basic information

Entry
Database: EMDB / ID: EMD-3291
TitleSub-tomogram averaging of Lassa virus glycoprotein spike from virus-like particles at pH 7
Map dataSub-tomogram average of the glycoprotein spike trimer
Sample
  • Sample: Purified Lassa virus VLPs at pH 7
  • Virus: Lassa virus
Keywordslassa virus / membrane protein / glycoprotein / receptor binding / membrane fusion
Biological speciesLassa virus
Methodsubtomogram averaging / cryo EM / Resolution: 13.9 Å
AuthorsLi S / Zhaoyang S / Pryce R / Parsy M-L / Fehling SK / Schlie K / Siebert CA / Garten W / Bowden TA / Strecker T / Huiskonen JT
CitationJournal: PLoS Pathog / Year: 2016
Title: Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.
Authors: Sai Li / Zhaoyang Sun / Rhys Pryce / Marie-Laure Parsy / Sarah K Fehling / Katrin Schlie / C Alistair Siebert / Wolfgang Garten / Thomas A Bowden / Thomas Strecker / Juha T Huiskonen /
Abstract: Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, ...Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.
History
DepositionJan 9, 2016-
Header (metadata) releaseJan 20, 2016-
Map releaseFeb 17, 2016-
UpdateMar 9, 2016-
Current statusMar 9, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.6
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3291.tif

Downloads & links

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Map

FileDownload / File: emd_3291.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSub-tomogram average of the glycoprotein spike trimer
Voxel sizeX=Y=Z: 2.7 Å
Density
Contour LevelBy AUTHOR: 1.6 / Movie #1: 1.6
Minimum - Maximum-3.62807751 - 6.55200863
Average (Standard dev.)0.02045275 (±0.54410708)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.72.72.7
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-3.6286.5520.020

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Supplemental data

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Sample components

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Entire : Purified Lassa virus VLPs at pH 7

EntireName: Purified Lassa virus VLPs at pH 7
Components
  • Sample: Purified Lassa virus VLPs at pH 7
  • Virus: Lassa virus

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Supramolecule #1000: Purified Lassa virus VLPs at pH 7

SupramoleculeName: Purified Lassa virus VLPs at pH 7 / type: sample / ID: 1000 / Details: Unfixed virus-like particles / Number unique components: 1

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Supramolecule #1: Lassa virus

SupramoleculeName: Lassa virus / type: virus / ID: 1 / Name.synonym: Lassa mammarenavirus / NCBI-ID: 11620 / Sci species name: Lassa virus / Sci species strain: Josiah / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No / Syn species name: Lassa mammarenavirus
Host (natural)Organism: Mastomys (multimammate rats) / synonym: VERTEBRATES
Host systemOrganism: Canis lupus (gray wolf) / Recombinant cell: Madin-Darby canine kidney

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.3
Details: 50 mM buffer of succinic acid, dihydrogen phosphate and glycine (2:7:7)
GridDetails: Grids (Cflat CF-2/1-2C-T) were glow-discharged for 15 s. 6-nm gold particles were added.
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 80 % / Chamber temperature: 120 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 3 seconds before plunging.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.7 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 160000
Specialist opticsEnergy filter - Name: GIF QUANTUM LS / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -45 ° / Tilt series - Axis1 - Max angle: 45 °
TemperatureMin: 80 K / Max: 120 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 160,000 times magnification.
DetailsSuper-resolution counting mode
DateMay 15, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 31 / Average electron dose: 60 e/Å2
Details: Each image is a tilt series of 19 movies, acquired at 5 degree intervals. Each movie consists of 8 frames.
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each tilted image
Final 3D classificationNumber classes: 1
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.9 Å / Resolution method: OTHER / Software - Name: IMOD, Dynamo / Number subtomograms used: 2764
DetailsSubtomograms were picked manually. No C3 symmetry was imposed in the initial stages of refinement.
FSC plot (resolution estimation)

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