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- EMDB-8714: Cryo-EM reconstruction of B41 SOSIP.664 -

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Basic information

Entry
Database: EMDB / ID: EMD-8714
TitleCryo-EM reconstruction of B41 SOSIP.664
Map dataCryo-EM reconstruction of B41 SOSIP.664
Sample
  • Complex: HIV-1 Env B41 SOSIP.664
    • Protein or peptide: HIV-1 Env B41 SOSIP.664 gp41
    • Protein or peptide: HIV-1 Env B41 SOSIP.664 gp120
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
Methodsingle particle reconstruction / cryo EM / Resolution: 5.6 Å
AuthorsPallesen J / Ozorowski G / de Val N / Ward AB
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) releaseJun 14, 2017-
Map releaseJul 26, 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: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8714.png

Downloads & links

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Map

FileDownload / File: emd_8714.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of B41 SOSIP.664
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 220 pix.
= 288.2 Å		1.31 Å/pix.
x 220 pix.
= 288.2 Å		1.31 Å/pix.
x 220 pix.
= 288.2 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: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.03967638 - 0.08950094
Average (Standard dev.)0.000056482935 (±0.0040740515)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 288.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z288.200288.200288.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ281156
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.0400.0900.000

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Supplemental data

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Additional map: Cryo-EM reconstruction of B41 SOSIP.664, additional map

Fileemd_8714_additional.map
AnnotationCryo-EM reconstruction of B41 SOSIP.664, additional map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_8714_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_8714_half_map_2.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

EntireName: HIV-1 Env B41 SOSIP.664
Components
  • Complex: HIV-1 Env B41 SOSIP.664
    • Protein or peptide: HIV-1 Env B41 SOSIP.664 gp41
    • Protein or peptide: HIV-1 Env B41 SOSIP.664 gp120

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

SupramoleculeName: HIV-1 Env B41 SOSIP.664 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: homo sapiens (human) / Recombinant cell: HEK293F
Molecular weightTheoretical: 480 KDa

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Macromolecule #1: HIV-1 Env B41 SOSIP.664 gp41

MacromoleculeName: HIV-1 Env B41 SOSIP.664 gp41 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: 9032-08.A1.4685
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
AVGLGA FIL GFLGAAG ST MGAASMAL T VQARLLLSG IVQQQNNLLR APEAQQHML Q LTVWGIKQ LQ ARVLAVE RYL RDQQLL GIWG CSGKI ICCTN VPWN DSWSNK TIN EIWDNMT WM QWEKEIDN Y TQHIYTLLE VSQIQQEKNE QELLELD

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Macromolecule #2: HIV-1 Env B41 SOSIP.664 gp120

MacromoleculeName: HIV-1 Env B41 SOSIP.664 gp120 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: 9032-08.A1.4685
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDAMKRGLCC VLLLCGAVF V SPSQEIHA RF RRGARAA KKW VTVYYG VPVW KEATT TLFCA SDAK AYDTEV HNV WATHACV PT DPNPQEIV L GNVTENFNM WKNNMVEQMH EDIISLWDQ S LKPCVKLT PL CVTLNCN NVN TNNTNN STNA TISDW ...String:
MDAMKRGLCC VLLLCGAVF V SPSQEIHA RF RRGARAA KKW VTVYYG VPVW KEATT TLFCA SDAK AYDTEV HNV WATHACV PT DPNPQEIV L GNVTENFNM WKNNMVEQMH EDIISLWDQ S LKPCVKLT PL CVTLNCN NVN TNNTNN STNA TISDW EKMET GEMK NCSFNV TTS IRDKIKK EY ALFYKLDV V PLENKNNIN NTNITNYRLI NCNTSVITQ A CPKVSFEP IP IHYCAPA GFA ILKCNS KTFN GSGPC TNVST VQCT HGIRPV VST QLLLNGS LA EEEIVIRS E NITDNAKTI IVQLNEAVEI NCTRPNNNT R KSIHIGPG RA FYATGDI IGN IRQAHC NISK ARWNE TLGQI VAKL EEQFPN KTI IFNHSSG GD PEIVTHSF N CGGEFFYCN TTPLFNSTWN NTRTDDYPT G GEQNITLQ CR IKQIINM WQG VGKAMY APPI RGQIR CSSNI TGLL LTRDGG RDQ NGTETFR PG GGNMRDNW R SELYKYKVV KIEPLGIAPT ACKRRVVQR RRR

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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:
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 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: 4.5 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: 1-35 / Number real images: 2042 / Average exposure time: 7.0 sec. / Average electron dose: 41.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND3
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: 1.88 degrees
Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 5.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 42541
FSC plot (resolution estimation)

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