|Entry||Database: EMDB / ID: 9061|
|Title||HA-GCN4pL-sfGFP map in negative stain EM|
|Map data||HA-GCN4pL-sfGFP from negative stain EM|
|Sample||3D map of HA-GCN4pL-sfGFP from negative stain EM:|
|Source||Influenza A virus|
|Method||single particle reconstruction / 20 Å resolution|
|Authors||Ward AB / Turner HL|
|Citation||Journal: 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.
|Date||Deposition: Aug 27, 2018 / Header (metadata) release: Sep 19, 2018 / Map release: Jan 16, 2019 / Last update: Jan 16, 2019|
|Structure viewer||EM map: |
Downloads & links
|File||emd_9061.map.gz (map file in CCP4 format, 44958 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.05 Å|
CCP4 map header:
-Entire 3D map of HA-GCN4pL-sfGFP from negative stain EM
|Entire||Name: 3D map of HA-GCN4pL-sfGFP from negative stain EM / Number of components: 1|
-Component #1: protein, 3D map of HA-GCN4pL-sfGFP from negative stain EM
|Protein||Name: 3D map of HA-GCN4pL-sfGFP from negative stain EM / Recombinant expression: No|
|Source||Species: Influenza A virus|
|Source (engineered)||Expression System: Homo sapiens (human)|
|Specimen||Specimen state: particle|
|Sample solution||pH: 7.4|
|Vitrification||Cryogen name: NONE|
-Electron microscopy imaging
Model: Tecnai Spirit / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI SPIRIT|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: -1500.0 - nm|
|Specimen Holder||Model: OTHER|
|Camera||Detector: TVIPS TEMCAM-F416 (4k x 4k)|
|Image acquisition||Sampling size: 15.6 microns|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 11358|
|3D reconstruction||Resolution: 2 Å / Resolution method: FSC 0.5 CUT-OFF|
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