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

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Basic information

Entry
Database: EMDB / ID: EMD-9061
TitleHA-GCN4pL-sfGFP map in negative stain EM
Map dataHA-GCN4pL-sfGFP from negative stain EM
Sample
  • Complex: 3D map of HA-GCN4pL-sfGFP from negative stain EM
Biological speciesInfluenza A virus
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsWard AB / Turner HL
CitationJournal: J Mol Biol / Year: 2019
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 / ...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.
History
DepositionAug 27, 2018-
Header (metadata) releaseSep 19, 2018-
Map releaseJan 16, 2019-
UpdateJan 16, 2019-
Current statusJan 16, 2019Processing site: RCSB / Status: Released

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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
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9061.png

Downloads & links

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Map

FileDownload / File: emd_9061.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHA-GCN4pL-sfGFP from negative stain EM
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 0.00549 / Movie #1: 0.00549
Minimum - Maximum-0.025048986 - 0.10110831
Average (Standard dev.)0.00005762574 (±0.002976045)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 459.19998 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: 3D map of HA-GCN4pL-sfGFP from negative stain EM

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Supramolecule #1: 3D map of HA-GCN4pL-sfGFP from negative stain EM

SupramoleculeName: 3D map of HA-GCN4pL-sfGFP from negative stain EM / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Influenza A virus
Recombinant expressionOrganism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
StainingType: NEGATIVE / Material: Uranyl Formate
GridDetails: unspecified

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus min: -1.5 µm
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 15.6 µm / Average electron dose: 25.0 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 11358
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3vun

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 11358

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