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- EMDB-8240: EBOV GP in complex with IgG c2G4 and c13C6 Fabs -

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Basic information

Entry
Database: EMDB / ID: EMD-8240
TitleEBOV GP in complex with IgG c2G4 and c13C6 Fabs
Map dataEBOV GP in complex with variable Fab domains of IgGs c2G4 and c13C6
Sample
  • Complex: Ebola virus trimeric surface glycoprotein in complex with IgG c2G4 and c13C6 Fabs
    • Protein or peptide: Ebola surface glycoprotein, GP1
    • Protein or peptide: Ebola surface glycoprotein, GP2
    • Protein or peptide: c2G4 variable Fab domain heavy chain
    • Protein or peptide: c2G4 variable Fab domain light chain
    • Protein or peptide: c13C6 variable Fab domain heavy chain
    • Protein or peptide: c13C6 variable Fab domain light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


host cell endoplasmic reticulum / viral budding from plasma membrane / clathrin-dependent endocytosis of virus by host cell / suppression by virus of host tetherin activity / host cell cytoplasm / entry receptor-mediated virion attachment to host cell / symbiont entry into host cell / membrane raft / fusion of virus membrane with host endosome membrane / viral envelope ...host cell endoplasmic reticulum / viral budding from plasma membrane / clathrin-dependent endocytosis of virus by host cell / suppression by virus of host tetherin activity / host cell cytoplasm / entry receptor-mediated virion attachment to host cell / symbiont entry into host cell / membrane raft / fusion of virus membrane with host endosome membrane / viral envelope / lipid binding / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell plasma membrane / virion membrane / extracellular region / identical protein binding
Similarity search - Function
Filoviruses glycoprotein, extracellular domain / Filoviruses glycoprotein / Filovirus glycoprotein
Similarity search - Domain/homology
Envelope glycoprotein
Similarity search - Component
Biological speciesZaire ebolavirus / Zaire ebolavirus (strain Mayinga-76) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsPallesen J / Murin CD / de Val N / Cottrell CA / Hastie KM / Turner HL / Fusco ML / Flyak AI / Zeitlin L / Crowe Jr JE ...Pallesen J / Murin CD / de Val N / Cottrell CA / Hastie KM / Turner HL / Fusco ML / Flyak AI / Zeitlin L / Crowe Jr JE / Andersen KG / Saphire EO / Ward AB
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI067927 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI1097 11 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI109762 United States
CitationJournal: Nat Microbiol / Year: 2016
Title: Structures of Ebola virus GP and sGP in complex with therapeutic antibodies.
Authors: Jesper Pallesen / Charles D Murin / Natalia de Val / Christopher A Cottrell / Kathryn M Hastie / Hannah L Turner / Marnie L Fusco / Andrew I Flyak / Larry Zeitlin / James E Crowe / Kristian ...Authors: Jesper Pallesen / Charles D Murin / Natalia de Val / Christopher A Cottrell / Kathryn M Hastie / Hannah L Turner / Marnie L Fusco / Andrew I Flyak / Larry Zeitlin / James E Crowe / Kristian G Andersen / Erica Ollmann Saphire / Andrew B Ward /
Abstract: The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little ...The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little is known regarding its structure or functional role. A minor product, resulting from transcriptional editing, is the transmembrane-anchored, trimeric viral surface glycoprotein (GP). GP mediates attachment to and entry into host cells, and is the intended target of antibody therapeutics. Because large portions of sequence are shared between GP and sGP, it has been hypothesized that sGP may potentially subvert the immune response or may contribute to pathogenicity. In this study, we present cryo-electron microscopy structures of GP and sGP in complex with GP-specific and GP/sGP cross-reactive antibodies undergoing human clinical trials. The structure of the sGP dimer presented here, in complex with both an sGP-specific antibody and a GP/sGP cross-reactive antibody, permits us to unambiguously assign the oligomeric arrangement of sGP and compare its structure and epitope presentation to those of GP. We also provide biophysical evaluation of naturally occurring GP/sGP mutations that fall within the footprints identified by our high-resolution structures. Taken together, our data provide a detailed and more complete picture of the accessible Ebolavirus glycoprotein landscape and a structural basis to evaluate patient and vaccine antibody responses towards differently structured products of the GP gene.
History
DepositionJul 18, 2016-
Header (metadata) releaseSep 7, 2016-
Map releaseSep 7, 2016-
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: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5kel
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8240.png

Downloads & links

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Map

FileDownload / File: emd_8240.map.gz / Format: CCP4 / Size: 55.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEBOV GP in complex with variable Fab domains of IgGs c2G4 and c13C6
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 244 pix.
= 319.64 Å		1.31 Å/pix.
x 244 pix.
= 319.64 Å		1.31 Å/pix.
x 244 pix.
= 319.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.044981815 - 0.102471
Average (Standard dev.)0.0000281551 (±0.0038259183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions244244244
Spacing244244244
CellA=B=C: 319.63998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z244244244
origin x/y/z0.0000.0000.000
length x/y/z319.640319.640319.640
α/β/γ90.00090.00090.000
start NX/NY/NZ-51-35-11
NX/NY/NZ11110799
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS244244244
D min/max/mean-0.0450.1020.000

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Supplemental data

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Additional map: EBOV GP in complex with variable Fab domains of IgGs c2G4 and c13C6

Fileemd_8240_additional_1.map
AnnotationEBOV GP in complex with variable Fab domains of IgGs c2G4 and c13C6
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: EBOV GP in complex with variable Fab domains of IgGs c2G4 and c13C6

Fileemd_8240_additional_2.map
AnnotationEBOV GP in complex with variable Fab domains of IgGs c2G4 and c13C6
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: EBOV GP in complex with variable Fab domains of IgGs c2G4 and c13C6

Fileemd_8240_additional_3.map
AnnotationEBOV GP in complex with variable Fab domains of IgGs c2G4 and c13C6
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ebola virus trimeric surface glycoprotein in complex with IgG c2G...

EntireName: Ebola virus trimeric surface glycoprotein in complex with IgG c2G4 and c13C6 Fabs
Components
  • Complex: Ebola virus trimeric surface glycoprotein in complex with IgG c2G4 and c13C6 Fabs
    • Protein or peptide: Ebola surface glycoprotein, GP1
    • Protein or peptide: Ebola surface glycoprotein, GP2
    • Protein or peptide: c2G4 variable Fab domain heavy chain
    • Protein or peptide: c2G4 variable Fab domain light chain
    • Protein or peptide: c13C6 variable Fab domain heavy chain
    • Protein or peptide: c13C6 variable Fab domain light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Ebola virus trimeric surface glycoprotein in complex with IgG c2G...

SupramoleculeName: Ebola virus trimeric surface glycoprotein in complex with IgG c2G4 and c13C6 Fabs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Zaire ebolavirus / Strain: Mayinga-76
Molecular weightExperimental: 540 KDa

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Macromolecule #1: Ebola surface glycoprotein, GP1

MacromoleculeName: Ebola surface glycoprotein, GP1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Zaire ebolavirus (strain Mayinga-76) / Strain: Mayinga-76
Molecular weightTheoretical: 50.974031 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString: IPLGVIHNST LQVSDVDKLV CRDKLSSTNQ LRSVGLNLEG NGVATDVPSA TKRWGFRSGV PPKVVNYEAG EWAENCYNLE IKKPDGSEC LPAAPDGIRG FPRCRYVHKV SGTGPCAGDF AFHKEGAFFL YDRLASTVIY RGTTFAEGVV AFLILPQAKK D FFSSHPLR ...String:
IPLGVIHNST LQVSDVDKLV CRDKLSSTNQ LRSVGLNLEG NGVATDVPSA TKRWGFRSGV PPKVVNYEAG EWAENCYNLE IKKPDGSEC LPAAPDGIRG FPRCRYVHKV SGTGPCAGDF AFHKEGAFFL YDRLASTVIY RGTTFAEGVV AFLILPQAKK D FFSSHPLR EPVNATEDPS SGYYSTTIRY QATGFGTNET EYLFEVDNLT YVQLESRFTP QFLLQLNETI YTSGKRSNTT GK LIWKVNP EIDTTIGEWA FWETKKNLTR KIRSEELSFT VVSNGAKNIS GQSPARTSSD PGTNTTTEDH KIMASENSSA MVQ VHSQGR EAAVSHLTTL ATISTSPQSL TTKPGPDNST HNTPVYKLDI SEATQVEQHH RRTDNDSTAS DTPSATTAAG PPKA ENTNT SKSTDFLDPA TTTSPQNHSE TAGNNNTHHQ DTGEESASSG KLGLITNTIA GVAGLITGGR RTRR

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Macromolecule #2: Ebola surface glycoprotein, GP2

MacromoleculeName: Ebola surface glycoprotein, GP2 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Zaire ebolavirus / Strain: Mayinga-76
Molecular weightTheoretical: 16.137119 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString:
EAIVNAQPKC NPNLHYWTTQ DEGAAIGLAW IPYFGPAAEG IYTEGLMHNQ DGLICGLRQL ANETTQALQL FLRATTELRT FSILNRKAI DFLLQRWGGT CHILGPDCCI EPHDWTKNIT DKIDQIIHDF VDKTLPDLEV DDDD

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Macromolecule #3: c2G4 variable Fab domain heavy chain

MacromoleculeName: c2G4 variable Fab domain heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.573105 KDa
Recombinant expressionOrganism: Nicotiana benthamiana (plant)
SequenceString:
EVQLQESGGG LMQPGGSMKL SCVASGFTFS NYWMNWVRQS PEKGLEWVAE IRLKSNNYAT HYAESVKGRF TISRDDSKRS VYLQMNTLR AEDTGIYYCT RGNGNYRAMD YWGQGTSVTV S

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Macromolecule #4: c2G4 variable Fab domain light chain

MacromoleculeName: c2G4 variable Fab domain light chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.641884 KDa
Recombinant expressionOrganism: Nicotiana benthamiana (plant)
SequenceString:
DIQMTQSPAS LSVSVGETVS ITCRASENIY SSLAWYQQKQ GKSPQLLVYS ATILADGVPS RFSGSGSGTQ YSLKINSLQS EDFGTYYCQ HFWGTPYTFG GGTKLEIK

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Macromolecule #5: c13C6 variable Fab domain heavy chain

MacromoleculeName: c13C6 variable Fab domain heavy chain / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.169679 KDa
Recombinant expressionOrganism: Nicotiana benthamiana (plant)
SequenceString:
DVKLLESGGG LVQPGGSLKL SCAASGFSLS TSGVGVGWFR QPSGKGLEWL ALIWWDDDKY YNPSLKSQLS ISKDFSRNQV FLKISNVDI ADTATYYCAR RDPFGYDNAM GYWGQGTSVT VS

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Macromolecule #6: c13C6 variable Fab domain light chain

MacromoleculeName: c13C6 variable Fab domain light chain / type: protein_or_peptide / ID: 6 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.611877 KDa
Recombinant expressionOrganism: Nicotiana benthamiana (plant)
SequenceString:
DIVMTQSPLS LSTSVGDRVS LTCKASQNVG TAVAWYQQKP GQSPKLLIYS ASNRYTGVPD RFTGSGSGTD FTLTISNMQS EDLADYFCQ QYSSYPLTFG AGTKLELR

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Macromolecule #11: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 11 / Number of copies: 3 / 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

Concentration4 mg/mL
BufferpH: 7.4 / Component - Name: 1xTBS
GridModel: C-flat-2/2 4C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.0093 kPa
VitrificationCryogen name: ETHANE / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 77.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2526 / Average exposure time: 10.0 sec. / Average electron dose: 57.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND3
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3csy


Details: Only GP1/GP2 residues were included in initial model generation.
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 7.5 degrees
Software - Name: RELION (ver. 1.4b1)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 1.4b1)
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 0.9375 degrees
Software - Name: RELION (ver. 1.4b1)
Final reconstructionNumber classes used: 6 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4b1) / Number images used: 86000
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel building and refinement were conducted using a combination of software programs. The refinement target was optimizing using the MolProbity score while maintaining a high (good) EMRinger score.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: EMRinger
Output model

PDB-5kel:
EBOV GP in complex with variable Fab domains of IgGs c2G4 and c13C6

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