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- EMDB-9061: HA-GCN4pL-sfGFP map in negative stain EM -

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Basic information

Entry
Database: EMDB / ID: 9061
TitleHA-GCN4pL-sfGFP map in negative stain EM
Map dataHA-GCN4pL-sfGFP from negative stain EM
Sample3D map of HA-GCN4pL-sfGFP from negative stain EM:
SourceInfluenza A virus
Methodsingle particle reconstruction / 20 Å resolution
AuthorsWard AB / Turner HL
CitationJournal: J. Mol. Biol. / Year: 2018
Title: Fluorescent Trimeric Hemagglutinins Reveal Multivalent Receptor Binding Properties.
Authors: Nikoloz Nemanichvili / Ilhan Tomris / Hannah L Turner / Ryan McBride / Oliver C Grant / Roosmarijn van der Woude / Mohammed H Aldosari / Roland J Pieters / Robert J Woods / James C Paulson / Geert-Jan Boons / Andrew B Ward / Monique H Verheije / Robert P de Vries
Abstract: Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a ...Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA-receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA-sfGFP proteins was studied using glycan arrays and tissue staining. The HA-sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA-sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.
DateDeposition: Aug 27, 2018 / Header (metadata) release: Sep 19, 2018 / Map release: Jan 16, 2019 / Last update: Jan 16, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00549
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.00549
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_9061.map.gz (map file in CCP4 format, 44958 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
224 pix
2.05 Å/pix.
= 459.2 Å
224 pix
2.05 Å/pix.
= 459.2 Å
224 pix
2.05 Å/pix.
= 459.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.05 Å
Density
Contour Level:0.00549 (by author), 0.00549 (movie #1):
Minimum - Maximum-0.025048986 - 0.10110831
Average (Standard dev.)0.00005762574 (0.002976045)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions224224224
Origin0.00.00.0
Limit223.0223.0223.0
Spacing224224224
CellA=B=C: 459.19998 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z459.200459.200459.200
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.0250.1010.000

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Supplemental data

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Sample components

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Entire 3D map of HA-GCN4pL-sfGFP from negative stain EM

EntireName: 3D map of HA-GCN4pL-sfGFP from negative stain EM / Number of components: 1

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Component #1: protein, 3D map of HA-GCN4pL-sfGFP from negative stain EM

ProteinName: 3D map of HA-GCN4pL-sfGFP from negative stain EM / Recombinant expression: No
SourceSpecies: Influenza A virus
Source (engineered)Expression System: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle
Sample solutionpH: 7.4
Support filmunspecified
VitrificationCryogen name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI SPIRIT
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: -1500.0 - nm
Specimen HolderModel: OTHER
CameraDetector: TVIPS TEMCAM-F416 (4k x 4k)

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Image acquisition

Image acquisitionSampling size: 15.6 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 11358
3D reconstructionResolution: 2 Å / Resolution method: FSC 0.5 CUT-OFF

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