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- EMDB-3086: negative stain EM of BG505 SOSIP.664 in complex with sCD4, 17b, a... -

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Basic information

Entry
Database: EMDB / ID: EMD-3086
Titlenegative stain EM of BG505 SOSIP.664 in complex with sCD4, 17b, and 8ANC195
Map datanegative stain EM single particle analysis. The particles were picked using EMAN2. CTF correction was done using EMAN2. 2D and 3D classification was done using Relion to sort particles. The refinement and gold-standard FSC calculation was done using Relion.
Sample
  • Sample: soluble HIV-1 Env trimer BG505 SOSIP.664 in complex with soluble CD4s (D1-D2), broadly neutralizing antibody 17b Fabs, and broadly neutralizing antibody 8ANC195 variant G32K5 Fabs
  • Protein or peptide: Soluble HIV-1 Env trimer BG505 SOSIP.664
  • Protein or peptide: Fab of broadly neutralizing antibody 17b
  • Protein or peptide: soluble CD4 (D1-D2 domains)
  • Protein or peptide: Fab of broadly neutralizing antibody 8ANC195 variant G52K5
KeywordsHIV-1 Env trimer / broadly neutralizing antibody
Function / homology
Function and homology information


helper T cell enhancement of adaptive immune response / interleukin-16 binding / interleukin-16 receptor activity / maintenance of protein location in cell / T cell selection / MHC class II protein binding / cellular response to granulocyte macrophage colony-stimulating factor stimulus / interleukin-15-mediated signaling pathway / positive regulation of monocyte differentiation / Nef Mediated CD4 Down-regulation ...helper T cell enhancement of adaptive immune response / interleukin-16 binding / interleukin-16 receptor activity / maintenance of protein location in cell / T cell selection / MHC class II protein binding / cellular response to granulocyte macrophage colony-stimulating factor stimulus / interleukin-15-mediated signaling pathway / positive regulation of monocyte differentiation / Nef Mediated CD4 Down-regulation / Alpha-defensins / positive regulation of kinase activity / regulation of T cell activation / T cell receptor complex / extracellular matrix structural constituent / Other interleukin signaling / enzyme-linked receptor protein signaling pathway / Translocation of ZAP-70 to Immunological synapse / Phosphorylation of CD3 and TCR zeta chains / Dectin-2 family / regulation of calcium ion transport / macrophage differentiation / Generation of second messenger molecules / T cell differentiation / PD-1 signaling / positive regulation of protein kinase activity / Binding and entry of HIV virion / coreceptor activity / positive regulation of calcium-mediated signaling / positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / protein tyrosine kinase binding / positive regulation of establishment of T cell polarity / positive regulation of interleukin-2 production / virus-mediated perturbation of host defense response / T cell activation / host cell endosome membrane / calcium-mediated signaling / Vpu mediated degradation of CD4 / clathrin-coated endocytic vesicle membrane / cell surface receptor protein tyrosine kinase signaling pathway / positive regulation of T cell activation / positive regulation of peptidyl-tyrosine phosphorylation / transmembrane signaling receptor activity / Cargo recognition for clathrin-mediated endocytosis / Downstream TCR signaling / virus receptor activity / signaling receptor activity / Clathrin-mediated endocytosis / MHC class II protein complex binding / clathrin-dependent endocytosis of virus by host cell / positive regulation of canonical NF-kappaB signal transduction / defense response to Gram-negative bacterium / adaptive immune response / positive regulation of MAPK cascade / positive regulation of viral entry into host cell / positive regulation of ERK1 and ERK2 cascade / cell surface receptor signaling pathway / viral protein processing / early endosome / cell adhesion / immune response / positive regulation of protein phosphorylation / membrane raft / fusion of virus membrane with host plasma membrane / endoplasmic reticulum lumen / external side of plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / lipid binding / structural molecule activity / virion attachment to host cell / endoplasmic reticulum membrane / protein kinase binding / host cell plasma membrane / virion membrane / positive regulation of DNA-templated transcription / enzyme binding / signal transduction / protein homodimerization activity / zinc ion binding / membrane / identical protein binding / plasma membrane
Similarity search - Function
CD4, extracellular / T cell CD4 receptor C-terminal region / CD4, extracellular / T cell CD4 receptor C terminal region / T-cell surface antigen CD4 / Immunoglobulin C2-set / Immunoglobulin C2-set domain / Immunoglobulin / Immunoglobulin domain / Envelope glycoprotein Gp160 ...CD4, extracellular / T cell CD4 receptor C-terminal region / CD4, extracellular / T cell CD4 receptor C terminal region / T-cell surface antigen CD4 / Immunoglobulin C2-set / Immunoglobulin C2-set domain / Immunoglobulin / Immunoglobulin domain / Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120 / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
T-cell surface glycoprotein CD4 / Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 16.8 Å
AuthorsScharf L / Wang H / Gao H / Chen S / McDowall A / Bjorkman P
CitationJournal: Cell / Year: 2015
Title: Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env.
Authors: Louise Scharf / Haoqing Wang / Han Gao / Songye Chen / Alasdair W McDowall / Pamela J Bjorkman /
Abstract: The HIV-1 envelope (Env) spike contains limited epitopes for broadly neutralizing antibodies (bNAbs); thus, most neutralizing antibodies are strain specific. The 8ANC195 epitope, defined by crystal ...The HIV-1 envelope (Env) spike contains limited epitopes for broadly neutralizing antibodies (bNAbs); thus, most neutralizing antibodies are strain specific. The 8ANC195 epitope, defined by crystal and electron microscopy (EM) structures of bNAb 8ANC195 complexed with monomeric gp120 and trimeric Env, respectively, spans the gp120 and gp41 Env subunits. To investigate 8ANC195's gp41 epitope at higher resolution, we solved a 3.58 Å crystal structure of 8ANC195 complexed with fully glycosylated Env trimer, revealing 8ANC195 insertion into a glycan shield gap to contact gp120 and gp41 glycans and protein residues. To determine whether 8ANC195 recognizes the CD4-bound open Env conformation that leads to co-receptor binding and fusion, one of several known conformations of virion-associated Env, we solved EM structures of an Env/CD4/CD4-induced antibody/8ANC195 complex. 8ANC195 binding partially closed the CD4-bound trimer, confirming structural plasticity of Env by revealing a previously unseen conformation. 8ANC195's ability to bind different Env conformations suggests advantages for potential therapeutic applications.
History
DepositionJul 9, 2015-
Header (metadata) releaseJul 22, 2015-
Map releaseSep 23, 2015-
UpdateSep 23, 2015-
Current statusSep 23, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0269
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0269
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a7x
  • Surface level: 0.0269
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3086.jpg

Downloads & links

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Map

FileDownload / File: emd_3086.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnegative stain EM single particle analysis. The particles were picked using EMAN2. CTF correction was done using EMAN2. 2D and 3D classification was done using Relion to sort particles. The refinement and gold-standard FSC calculation was done using Relion.
Voxel sizeX=Y=Z: 2.5 Å
Density
Contour LevelBy AUTHOR: 0.0269 / Movie #1: 0.0269
Minimum - Maximum-0.07877693 - 0.0897764
Average (Standard dev.)-0.00034449 (±0.00837527)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-21-120
NX/NY/NZ432573
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0790.090-0.000

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Supplemental data

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Sample components

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Entire : soluble HIV-1 Env trimer BG505 SOSIP.664 in complex with soluble ...

EntireName: soluble HIV-1 Env trimer BG505 SOSIP.664 in complex with soluble CD4s (D1-D2), broadly neutralizing antibody 17b Fabs, and broadly neutralizing antibody 8ANC195 variant G32K5 Fabs
Components
  • Sample: soluble HIV-1 Env trimer BG505 SOSIP.664 in complex with soluble CD4s (D1-D2), broadly neutralizing antibody 17b Fabs, and broadly neutralizing antibody 8ANC195 variant G32K5 Fabs
  • Protein or peptide: Soluble HIV-1 Env trimer BG505 SOSIP.664
  • Protein or peptide: Fab of broadly neutralizing antibody 17b
  • Protein or peptide: soluble CD4 (D1-D2 domains)
  • Protein or peptide: Fab of broadly neutralizing antibody 8ANC195 variant G52K5

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Supramolecule #1000: soluble HIV-1 Env trimer BG505 SOSIP.664 in complex with soluble ...

SupramoleculeName: soluble HIV-1 Env trimer BG505 SOSIP.664 in complex with soluble CD4s (D1-D2), broadly neutralizing antibody 17b Fabs, and broadly neutralizing antibody 8ANC195 variant G32K5 Fabs
type: sample / ID: 1000
Oligomeric state: 3 Fabs of 17b, 3 sCD4, and 3 Fabs of 8ANC195 G52K5 bind to Env trimer BG505 SOSIP
Number unique components: 4
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: Soluble HIV-1 Env trimer BG505 SOSIP.664

MacromoleculeName: Soluble HIV-1 Env trimer BG505 SOSIP.664 / type: protein_or_peptide / ID: 1 / Name.synonym: BG505 SOSIP / Number of copies: 1 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / synonym: HIV-1
Molecular weightTheoretical: 420 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293-6E / Recombinant plasmid: pTT5

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Macromolecule #2: Fab of broadly neutralizing antibody 17b

MacromoleculeName: Fab of broadly neutralizing antibody 17b / type: protein_or_peptide / ID: 2 / Name.synonym: 17b / Number of copies: 3 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 50 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293-6E / Recombinant plasmid: pTT5

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Macromolecule #3: soluble CD4 (D1-D2 domains)

MacromoleculeName: soluble CD4 (D1-D2 domains) / type: protein_or_peptide / ID: 3 / Name.synonym: sCD4 / Number of copies: 3 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 20 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293-6E / Recombinant plasmid: pTT5

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Macromolecule #4: Fab of broadly neutralizing antibody 8ANC195 variant G52K5

MacromoleculeName: Fab of broadly neutralizing antibody 8ANC195 variant G52K5
type: protein_or_peptide / ID: 4 / Name.synonym: 8ANC195 G52K5 / Number of copies: 3 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 50 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293-6E / Recombinant plasmid: pTT5

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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

Concentration0.01 mg/mL
BufferpH: 8 / Details: 20mM Tris, 50mM NaCl
StainingType: NEGATIVE
Details: Grids with adsorbed protein cross-linked by glutaraldehyde vapor and stained by 3% w/v uranyl acetate for 30 seconds.
GridDetails: glow discharged ultrathin C film on holey carbon support film, 400 mesh, Cu grids
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal magnification: 42000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateApr 7, 2015
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Number real images: 642

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 25
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.8 Å / Resolution method: OTHER / Software - Name: EMAN2, Relion / Number images used: 7174
DetailsParticle were picked using EMAN2. CTF correction was done using EMAN2. 2D and 3D classification done using Relion to select good particles. Refinement done using Relion.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1rzk


Chain - #0 - Chain ID: G / Chain - #1 - Chain ID: C / Chain - #2 - Chain ID: L / Chain - #3 - Chain ID: H
SoftwareName: Chimera
DetailsFitted by manual docking first and then using Chimera with the following options: real-time correlation/average update, and use map simulated from atoms, resolution 15 A
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5a7x:
negative stain EM of BG505 SOSIP.664 in complex with sCD4, 17b, and 8ANC195

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Atomic model buiding 2

Initial modelPDB ID:

4p9m


Chain - #0 - Chain ID: L / Chain - #1 - Chain ID: H
SoftwareName: Chimera
DetailsFitted by manual docking first and then using Chimera with the following options: real-time correlation/average update, and use map simulated from atoms, resolution 15 A
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5a7x:
negative stain EM of BG505 SOSIP.664 in complex with sCD4, 17b, and 8ANC195

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