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- EMDB-8563: CryoEM structure of an influenza virus receptor-binding site anti... -

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Basic information

Entry
Database: EMDB / ID: EMD-8563
TitleCryoEM structure of an influenza virus receptor-binding site antibody-antigen interface - Class 3
Map datainfluenza virus receptor-binding site antibody-antigen interface - Class 3
Sample
  • Complex: Influenza-virus hemagglutinin H1 complexed with K1915 single-chain variable-domain fragment
    • Protein or peptide: Hemagglutinin HA1
    • Protein or peptide: Hemagglutinin HA2
    • Protein or peptide: scFvSingle-chain variable fragment
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


viral budding from plasma membrane / 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 / virion 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)) / Influenza A virus / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsLiu Y / Pan J / Caradonna T / Jenni S / Raymond DD / Schmidt AG / Harrison SC / Grigorieff N
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01 AI-089618 United States
CitationJournal: J Mol Biol / Year: 2017
Title: CryoEM Structure of an Influenza Virus Receptor-Binding Site Antibody-Antigen Interface.
Authors: Yuhang Liu / Junhua Pan / Simon Jenni / Donald D Raymond / Tim Caradonna / Khoi T Do / Aaron G Schmidt / Stephen C Harrison / Nikolaus Grigorieff /
Abstract: Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes.Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray ...Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes.Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structure of influenza-virus hemagglutinin (HA):single-chain variable-domain fragment complexes, by studying a complex we failed to crystallize in pursuing an extended project on the human immune response to influenza vaccines.The result shows that a combination of cryoEM and molecular modeling can yield details of the antigen-antibody interface, although small variation in the twist of the rod-likeHA trimer limited the overall resolution to about 4.5Å.Comparison of principal 3D classes suggests ways to modify the HA trimer to overcome this limitation. A closely related antibody from the same donor did yield crystals when bound with the same HA, giving us an independent validation of the cryoEM results.The two structures also augment our understanding of receptor-binding site recognition by antibodies that neutralize a wide range of influenza-virus variants.
History
DepositionJan 19, 2017-
Header (metadata) releaseFeb 15, 2017-
Map releaseMay 31, 2017-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5uk1
  • Surface level: 1.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8563.png

Downloads & links

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Map

FileDownload / File: emd_8563.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationinfluenza virus receptor-binding site antibody-antigen interface - Class 3
Voxel sizeX=Y=Z: 1.64 Å
Density
Contour LevelBy EMDB: 1.6 / Movie #1: 1.5
Minimum - Maximum-2.7165923 - 5.834584
Average (Standard dev.)0.06930954 (±0.3747907)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-60
Dimensions120120120
Spacing120120120
CellA=B=C: 196.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.641.641.64
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z196.800196.800196.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-60-60-60
NC/NR/NS120120120
D min/max/mean-2.7175.8350.069

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Supplemental data

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Sample components

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Entire : Influenza-virus hemagglutinin H1 complexed with K1915 single-chai...

EntireName: Influenza-virus hemagglutinin H1 complexed with K1915 single-chain variable-domain fragment
Components
  • Complex: Influenza-virus hemagglutinin H1 complexed with K1915 single-chain variable-domain fragment
    • Protein or peptide: Hemagglutinin HA1
    • Protein or peptide: Hemagglutinin HA2
    • Protein or peptide: scFvSingle-chain variable fragment
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Influenza-virus hemagglutinin H1 complexed with K1915 single-chai...

SupramoleculeName: Influenza-virus hemagglutinin H1 complexed with K1915 single-chain variable-domain fragment
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: The complex consists of three hemagglutinin head domains bound to three single-chain variable-domain fragments.
Source (natural)Organism: Influenza A virus (A/Solomon Islands/3/2006(H1N1))
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: Hi-5
Molecular weightTheoretical: 320 KDa

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Macromolecule #1: Hemagglutinin HA1

MacromoleculeName: Hemagglutinin HA1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (A/Solomon Islands/3/2006(H1N1))
Strain: A/Solomon Islands/3/2006(H1N1)
Molecular weightTheoretical: 36.024344 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: EDTICIGYHA NNSTDTVDTV LEKNVTVTHS VNLLEDSHNG KLCLLKGIAP LQLGNCSVAG WILGNPECEL LISRESWSYI VEKPNPENG TCYPGHFADY EELREQLSSV SSFERFEIFP KESSWPNHTT TGVSASCSHN GESSFYKNLL WLTGKNGLYP N LSKSYANN ...String:
EDTICIGYHA NNSTDTVDTV LEKNVTVTHS VNLLEDSHNG KLCLLKGIAP LQLGNCSVAG WILGNPECEL LISRESWSYI VEKPNPENG TCYPGHFADY EELREQLSSV SSFERFEIFP KESSWPNHTT TGVSASCSHN GESSFYKNLL WLTGKNGLYP N LSKSYANN KEKEVLVLWG VHHPPNIGDQ RALYHTENAY VSVVSSHYSR KFTPEIAKRP KVRDREGRIN YYWTLLEPGD TI IFEANGN LIAPRYAFAL SRGFGSGIIN SNAPMDECDA KCQTPQGAIN SSLPFQNVHP VTIGECPKYV RSAKLRMVTG LRN IPS

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Macromolecule #2: Hemagglutinin HA2

MacromoleculeName: Hemagglutinin HA2 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus / Strain: A/Solomon Islands/3/2006(H1N1)
Molecular weightTheoretical: 19.841041 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
GLFGAIAGFI EGGWTGMVDG WYGYHHQNEQ GSGYAADQKS TQNAINGITN KVNSVIEKMN TQFTAVGKEF NKLERRMENL NKKVDDGFI DIWTYNAELL VLLENERTLD FHDSNVKNLY EKVKSQLKNN AKEIGNGCFE FYHKCNDECM ESVKNGTYDY P KYSEESKL NREKI

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Macromolecule #3: scFv

MacromoleculeName: scFv / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.629385 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EIVLTQSPGT LSLSPGERAT LSCRASQSIS GYYLTWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYC QQYGISPVIT FGGGTNVEIK GGGGGSGGGG SGGGGSEVQL VESGGGLVQP GGSLRLSCAA SGFTFNIYDM H WVRQAPGK ...String:
EIVLTQSPGT LSLSPGERAT LSCRASQSIS GYYLTWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYC QQYGISPVIT FGGGTNVEIK GGGGGSGGGG SGGGGSEVQL VESGGGLVQP GGSLRLSCAA SGFTFNIYDM H WVRQAPGK GLEWVSGLTT GGDTSYSGSV RGRFSISREN AKNSLYLQMN NLRAGDTAAY FCVRGVREVG ATGGDPFYYA MA VWGQGTT VTVSSASGSS GSGHHHHHH

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 18 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 8
Details: Beta-octylglucoside was added to a final concentration of 0.07% w/v to induce more variable particle orientations.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 9.0 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 30444 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 18000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Sampling interval: 2.5 µm / Digitization - Frames/image: 1-38 / Number grids imaged: 1 / Number real images: 10281 / Average exposure time: 13.0 sec. / Average electron dose: 40.0 e/Å2
Details: The exposure rate was 8 electrons/physical pixel/second.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 252130
CTF correctionSoftware - Name: CTFFIND3
Startup modelType of model: OTHER
Details: A starting map was determined using rosettes formed from Ni-NTA nanogold and His-tagged particles.
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 7.5 degrees
Software - Name: IMAGIC
Final 3D classificationNumber classes: 4 / Avg.num./class: 35579 / Software - Name: FREALIGN (ver. 9.11)
Details: Three out of the four classes showed high-resolution features.
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11)
Final reconstructionNumber classes used: 3 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11)
Details: Refinement and classification were limited to 10 Angstrom resolution.
Number images used: 142314
DetailsThe particle images were normalized to have constant variance and zero average. Movies were processed using Unblur.
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsDomains were initially placed manually using Chimera. The HA trimer was from PDB ID 5UGY. Fab was initially obtained using MODELLER, with PDB ID 4K8R as template. The structure was refined using PHENIX.
RefinementSpace: REAL / Protocol: OTHER / Target criteria: Correlation coefficient
Output model

PDB-5uk1:
CryoEM structure of an influenza virus receptor-binding site antibody-antigen interface - Class 3

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