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- EMDB-8717: Cryo-EM reconstruction of B41 SOSIP.664 in complex with fragment ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8717
TitleCryo-EM reconstruction of B41 SOSIP.664 in complex with fragment antigen binding of b12
Map dataCryo-EM model of B41 SOSIP.664 in complex with fragment antigen binding variable domain of b12
Sample
  • Complex: HIV-1 Env B41 SOSIP.664 in complex with b12 fragment antigen binding
    • Complex: HIV-1 Env B41 SOSIP.664
      • Protein or peptide: Envelope glycoprotein gp160
      • Protein or peptide: Envelope glycoprotein gp160
    • Complex: b12 fragment
      • Protein or peptide: b12 Fab heavy chain
      • Protein or peptide: b12 Fab light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / virus-mediated perturbation of host defense response / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / virus-mediated perturbation of host defense response / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / structural molecule activity / virion attachment to host cell / host cell plasma membrane / virion membrane / plasma membrane
Similarity search - Function
Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120
Similarity search - Domain/homology
Envelope glycoprotein gp160 / Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsPallesen J / Ozorowski G / de Val N / Ward AB
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM1AI100663 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01 AI110657 United States
CitationJournal: Nature / Year: 2017
Title: Open and closed structures reveal allostery and pliability in the HIV-1 envelope spike.
Authors: Gabriel Ozorowski / Jesper Pallesen / Natalia de Val / Dmitry Lyumkis / Christopher A Cottrell / Jonathan L Torres / Jeffrey Copps / Robyn L Stanfield / Albert Cupo / Pavel Pugach / John P ...Authors: Gabriel Ozorowski / Jesper Pallesen / Natalia de Val / Dmitry Lyumkis / Christopher A Cottrell / Jonathan L Torres / Jeffrey Copps / Robyn L Stanfield / Albert Cupo / Pavel Pugach / John P Moore / Ian A Wilson / Andrew B Ward /
Abstract: For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for ...For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. Although Env can tolerate a high degree of mutation in five variable regions (V1-V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env, and are targets of broadly neutralizing antibodies. Thus, they are attractive immunogens for vaccine development. Here we present high-resolution cryo-electron microscopy structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of 3.7 Å and 3.6 Å, respectively. We compare these to cryo-electron microscopy reconstructions of B41 SOSIP Env trimers with no ligand or in complex with either CD4 or the CD4-binding-site antibody PGV04 at 5.6 Å, 5.2 Å and 7.4 Å resolution, respectively. Consequently, we present the most complete description yet, to our knowledge, of the CD4-17b-induced intermediate and provide the molecular basis of the receptor-binding-induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.
History
DepositionApr 28, 2017-
Header (metadata) releaseJul 12, 2017-
Map releaseJul 12, 2017-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5vn8
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8717.png

Downloads & links

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Map

FileDownload / File: emd_8717.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM model of B41 SOSIP.664 in complex with fragment antigen binding variable domain of b12
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 256 pix.
= 335.36 Å		1.31 Å/pix.
x 256 pix.
= 335.36 Å		1.31 Å/pix.
x 256 pix.
= 335.36 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: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.20810786 - 0.39907405
Average (Standard dev.)0.00026210453 (±0.010594209)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z335.360335.360335.360
α/β/γ90.00090.00090.000
start NX/NY/NZ00-41
NX/NY/NZ737384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2080.3990.000

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Supplemental data

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Additional map: Cryo-EM model of B41 SOSIP.664 in complex with...

Fileemd_8717_additional.map
AnnotationCryo-EM model of B41 SOSIP.664 in complex with fragment antigen binding variable domain of b12, additional map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM model of B41 SOSIP.664 in complex with...

Fileemd_8717_half_map_1.map
AnnotationCryo-EM model of B41 SOSIP.664 in complex with fragment antigen binding variable domain of b12, half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM model of B41 SOSIP.664 in complex with...

Fileemd_8717_half_map_2.map
AnnotationCryo-EM model of B41 SOSIP.664 in complex with fragment antigen binding variable domain of b12, half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : HIV-1 Env B41 SOSIP.664 in complex with b12 fragment antigen binding

EntireName: HIV-1 Env B41 SOSIP.664 in complex with b12 fragment antigen binding
Components
  • Complex: HIV-1 Env B41 SOSIP.664 in complex with b12 fragment antigen binding
    • Complex: HIV-1 Env B41 SOSIP.664
      • Protein or peptide: Envelope glycoprotein gp160
      • Protein or peptide: Envelope glycoprotein gp160
    • Complex: b12 fragment
      • Protein or peptide: b12 Fab heavy chain
      • Protein or peptide: b12 Fab light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: HIV-1 Env B41 SOSIP.664 in complex with b12 fragment antigen binding

SupramoleculeName: HIV-1 Env B41 SOSIP.664 in complex with b12 fragment antigen binding
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Molecular weightTheoretical: 570 KDa

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Supramolecule #2: HIV-1 Env B41 SOSIP.664

SupramoleculeName: HIV-1 Env B41 SOSIP.664 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F

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Supramolecule #3: b12 fragment

SupramoleculeName: b12 fragment / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F

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Macromolecule #1: Envelope glycoprotein gp160

MacromoleculeName: Envelope glycoprotein gp160 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 57.702469 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDAMKRGLCC VLLLCGAVFV SPSQEIHARF RRGARAAKKW VTVYYGVPVW KEATTTLFCA SDAKAYDTEV HNVWATHACV PTDPNPQEI VLGNVTENFN MWKNNMVEQM HEDIISLWDQ SLKPCVKLTP LCVTLNCNNV NTNNTNNSTN ATISDWEKME T GEMKNCSF ...String:
MDAMKRGLCC VLLLCGAVFV SPSQEIHARF RRGARAAKKW VTVYYGVPVW KEATTTLFCA SDAKAYDTEV HNVWATHACV PTDPNPQEI VLGNVTENFN MWKNNMVEQM HEDIISLWDQ SLKPCVKLTP LCVTLNCNNV NTNNTNNSTN ATISDWEKME T GEMKNCSF NVTTSIRDKI KKEYALFYKL DVVPLENKNN INNTNITNYR LINCNTSVIT QACPKVSFEP IPIHYCAPAG FA ILKCNSK TFNGSGPCTN VSTVQCTHGI RPVVSTQLLL NGSLAEEEIV IRSENITDNA KTIIVQLNEA VEINCTRPNN NTR KSIHIG PGRAFYATGD IIGNIRQAHC NISKARWNET LGQIVAKLEE QFPNKTIIFN HSSGGDPEIV THSFNCGGEF FYCN TTPLF NSTWNNTRTD DYPTGGEQNI TLQCRIKQII NMWQGVGKAM YAPPIRGQIR CSSNITGLLL TRDGGRDQNG TETFR PGGG NMRDNWRSEL YKYKVVKIEP LGIAPTACKR RV

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Macromolecule #2: Envelope glycoprotein gp160

MacromoleculeName: Envelope glycoprotein gp160 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 17.357824 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
AVGLGAFILG FLGAAGSTMG AASMALTVQA RLLLSGIVQQ QNNLLRAPEA QQHMLQLTVW GIKQLQARVL AVERYLRDQQ LLGIWGCSG KIICCTNVPW NDSWSNKTIN EIWDNMTWMQ WEKEIDNYTQ HIYTLLEVSQ IQQEKNEQEL LELD

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Macromolecule #3: b12 Fab heavy chain

MacromoleculeName: b12 Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.910846 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLVQSGAE VKKPGASVKV SCQASGYRFS NFVIHWVRQA PGQRFEWMGW INPYNGNKEF SAKFQDRVTF TADTSANTAY MELRSLRSA DTAVYYCARV GPYSWDDSPQ DNYYMDVWGK GTTVIVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String:
QVQLVQSGAE VKKPGASVKV SCQASGYRFS NFVIHWVRQA PGQRFEWMGW INPYNGNKEF SAKFQDRVTF TADTSANTAY MELRSLRSA DTAVYYCARV GPYSWDDSPQ DNYYMDVWGK GTTVIVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SC

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Macromolecule #4: b12 Fab light chain

MacromoleculeName: b12 Fab light chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.707354 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EIVLTQSPGT LSLSPGERAT FSCRSSHSIR SRRVAWYQHK PGQAPRLVIH GVSNRASGIS DRFSGSGSGT DFTLTITRVE PEDFALYYC QVYGASSYTF GQGTKLERKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
EIVLTQSPGT LSLSPGERAT FSCRSSHSIR SRRVAWYQHK PGQAPRLVIH GVSNRASGIS DRFSGSGSGT DFTLTITRVE PEDFALYYC QVYGASSYTF GQGTKLERKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLRSPVTK SFNRGEC

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 33 / 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

Concentration5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
50.0 mMTris
0.06 mMDDM

Details: DDM was added to a final concentration of 0.06 mM prior to vitrification
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: 3 uL of sample applied to a holey carbon grid on glow discharged face and blotted manually on sample side until filter paper detached from grid, followed by immediate plunging.
DetailsB41 SOSIP.664 was incubated with a 6X molar excess of 17b Fab overnight at room temperature, purified by size exclusion chromatography, and concentrated prior to grid application

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 38168 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 0.000131 µm / Digitization - Frames/image: 1-50 / Number real images: 1300 / Average exposure time: 10.0 sec. / Average electron dose: 58.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.06)
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 7.5 degrees
Software - Name: RELION (ver. 2.0)
Final 3D classificationSoftware - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 0.94 degrees
Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 88071
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsHomology model of B41 SOSIP.664 created using PDB 5CEZ as initial model. b12 coordinates taken from PDB 2NY7. Performed fragment based refinement using Rosetta, followed by relaxed refinement in Rosetta. Modeled glycans using Chimera and performed final refinements using Phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: EMRinger
Output model

PDB-5vn8:
Cryo-EM model of B41 SOSIP.664 in complex with fragment antigen binding variable domain of b12

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