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- EMDB-8578: Negative Stain EM of C05 mutant Fab (VPGSGW) and CR9114 Fab in co... -

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Basic information

Entry
Database: EMDB / ID: EMD-8578
TitleNegative Stain EM of C05 mutant Fab (VPGSGW) and CR9114 Fab in complex with H1 HA Trimer
Map dataEM negative stain map of CR9114 and VPGSGW fabs in complex with H1 hemagglutinin trimer.
Sample
  • Complex: Negative stain EM map of C05 Mutant Fab (VPGSGW) and CR9114 fabs in complex with H1 HA trimer.
Function / homology
Function and homology information


clathrin-dependent endocytosis of virus by host cell / host cell surface receptor binding / apical plasma membrane / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane
Similarity search - Function
Haemagglutinin, influenzavirus A / Haemagglutinin, HA1 chain, alpha/beta domain superfamily / Haemagglutinin / Haemagglutinin, influenzavirus A/B / Viral capsid/haemagglutinin protein
Similarity search - Domain/homology
Biological speciesInfluenza A virus (A/Solomon Islands/3/2006(H1N1))
Methodsingle particle reconstruction / negative staining / Resolution: 18.0 Å
AuthorsTurner HL / Ward AB
CitationJournal: Nat Commun / Year: 2017
Title: In vitro evolution of an influenza broadly neutralizing antibody is modulated by hemagglutinin receptor specificity.
Authors: Nicholas C Wu / Geramie Grande / Hannah L Turner / Andrew B Ward / Jia Xie / Richard A Lerner / Ian A Wilson /
Abstract: The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. ...The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs. The C05 variants evolve up to 20-fold higher affinity but increase specificity to each HA subtype used in the selection. Structural analysis reveals that the fine specificity is strongly influenced by a highly conserved substitution that regulates receptor binding in different subtypes. Overall, this study suggests that subtle natural variations in the HA RBS between subtypes and species may differentially influence the evolution of high-affinity bnAbs.
History
DepositionFeb 1, 2017-
Header (metadata) releaseFeb 15, 2017-
Map releaseMay 31, 2017-
UpdateFeb 14, 2018-
Current statusFeb 14, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 20.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 20.7
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8578.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM negative stain map of CR9114 and VPGSGW fabs in complex with H1 hemagglutinin trimer.
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 20.699999999999999 / Movie #1: 20.7
Minimum - Maximum-48.753773000000002 - 100.832436000000001
Average (Standard dev.)0.7061781 (±8.199496)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 295.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z295.200295.200295.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ281156
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-48.754100.8320.706

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Supplemental data

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Sample components

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Entire : Negative stain EM map of C05 Mutant Fab (VPGSGW) and CR9114 fabs ...

EntireName: Negative stain EM map of C05 Mutant Fab (VPGSGW) and CR9114 fabs in complex with H1 HA trimer.
Components
  • Complex: Negative stain EM map of C05 Mutant Fab (VPGSGW) and CR9114 fabs in complex with H1 HA trimer.

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Supramolecule #1: Negative stain EM map of C05 Mutant Fab (VPGSGW) and CR9114 fabs ...

SupramoleculeName: Negative stain EM map of C05 Mutant Fab (VPGSGW) and CR9114 fabs in complex with H1 HA trimer.
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Influenza A virus (A/Solomon Islands/3/2006(H1N1))
Strain: A/Solomon Islands/3/2006(H1N1)
Recombinant expressionOrganism: Drosophila (fruit flies)

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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

Concentration0.019 mg/mL
BufferpH: 7.4 / Component - Name: Tris Buffer Saline / Details: Solution was made fresh using 0.2um filter.
StainingType: NEGATIVE / Material: Uranyl Formate
Details: 0.2% Uranyl Formate. Samples were placed on 400 mesh copper grids covered with nitrocellulose. Samples were blotted off and UF was added for a total of 30 seconds before being blotted off.
GridModel: EMS / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 93 / 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: 18874
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: SPARX
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: sxali3d.py / Number images used: 18874

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