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- EMDB-20273: Negative-stain reconstruction of HIV-1 Env BG505 NFL CC+ trimer i... -

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Basic information

Entry
Database: EMDB / ID: EMD-20273
TitleNegative-stain reconstruction of HIV-1 Env BG505 NFL CC+ trimer in complex with rabbit antibody E70 fragment antigen binding
Map datanegative-stain 3D reconstruction
Sample
  • Complex: HIV-1 Env BG505 NFL CC+ trimer in complex with rabbit antibody E70 fragment antigen binding
    • Protein or peptide: HIV-1 Env BG505 NFL CC+ trimer
    • Protein or peptide: Rabbit antibody E70 fragment antigen binding heavy chain
    • Protein or peptide: Rabbit antibody E70 fragment antigen binding kappa chain
Biological speciesHuman immunodeficiency virus 1 / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsTorres JL / Ozorowski G / Andrew AB
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesUM AI100663 United States
Bill & Melinda Gates FoundationOPP1084519 United States
Bill & Melinda Gates FoundationOPP1115782 United States
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesAI136621 United States
CitationJournal: Immunity / Year: 2019
Title: Vaccination with Glycan-Modified HIV NFL Envelope Trimer-Liposomes Elicits Broadly Neutralizing Antibodies to Multiple Sites of Vulnerability.
Authors: Viktoriya Dubrovskaya / Karen Tran / Gabriel Ozorowski / Javier Guenaga / Richard Wilson / Shridhar Bale / Christopher A Cottrell / Hannah L Turner / Gemma Seabright / Sijy O'Dell / Jonathan ...Authors: Viktoriya Dubrovskaya / Karen Tran / Gabriel Ozorowski / Javier Guenaga / Richard Wilson / Shridhar Bale / Christopher A Cottrell / Hannah L Turner / Gemma Seabright / Sijy O'Dell / Jonathan L Torres / Lifei Yang / Yu Feng / Daniel P Leaman / Néstor Vázquez Bernat / Tyler Liban / Mark Louder / Krisha McKee / Robert T Bailer / Arlette Movsesyan / Nicole A Doria-Rose / Marie Pancera / Gunilla B Karlsson Hedestam / Michael B Zwick / Max Crispin / John R Mascola / Andrew B Ward / Richard T Wyatt /
Abstract: The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are ...The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination.
History
DepositionJun 10, 2019-
Header (metadata) releaseJul 3, 2019-
Map releaseNov 20, 2019-
UpdateApr 1, 2020-
Current statusApr 1, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20273.png

Downloads & links

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Map

FileDownload / File: emd_20273.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnegative-stain 3D reconstruction
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.02013376 - 0.089495584
Average (Standard dev.)0.00027650956 (±0.0054286076)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z328.000328.000328.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.0200.0890.000

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Supplemental data

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Half map: half map 1

Fileemd_20273_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_20273_half_map_2.map
Annotationhalf map 2
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 BG505 NFL CC+ trimer in complex with rabbit antibody E7...

EntireName: HIV-1 Env BG505 NFL CC+ trimer in complex with rabbit antibody E70 fragment antigen binding
Components
  • Complex: HIV-1 Env BG505 NFL CC+ trimer in complex with rabbit antibody E70 fragment antigen binding
    • Protein or peptide: HIV-1 Env BG505 NFL CC+ trimer
    • Protein or peptide: Rabbit antibody E70 fragment antigen binding heavy chain
    • Protein or peptide: Rabbit antibody E70 fragment antigen binding kappa chain

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Supramolecule #1: HIV-1 Env BG505 NFL CC+ trimer in complex with rabbit antibody E7...

SupramoleculeName: HIV-1 Env BG505 NFL CC+ trimer in complex with rabbit antibody E70 fragment antigen binding
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 570 KDa

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Macromolecule #1: HIV-1 Env BG505 NFL CC+ trimer

MacromoleculeName: HIV-1 Env BG505 NFL CC+ trimer / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPMGSLQPLA TLYLLGMLVA SVLAAENLWV TVYYGVPVWK DAETTLFCAS DAKAYETEKH NVWATHACVP TDPNPQEIHL ENVTEEFNMW KNNMVEQMHT DIISLWDQSL KPCVKLTPLC VTLQCTNVTN NITDDMRGEL KNCSFNMTTE LRDKKQKVYS LFYRLDVVQI ...String:
MPMGSLQPLA TLYLLGMLVA SVLAAENLWV TVYYGVPVWK DAETTLFCAS DAKAYETEKH NVWATHACVP TDPNPQEIHL ENVTEEFNMW KNNMVEQMHT DIISLWDQSL KPCVKLTPLC VTLQCTNVTN NITDDMRGEL KNCSFNMTTE LRDKKQKVYS LFYRLDVVQI NENQGNRSNN SNKEYRLINC NTSACTQACP KVSFEPIPIH YCAPAGFAIL KCKDKKFNGT GPCPSVSTVQ CTHGIKPVVS TQLLLNGSLA EEEVMIRSEN ITNNAKNILV QFNTPVQINC TRPNNYTRKS IRIGPGQAFY ATGDIIGDIR QAHCNVSKAT WNETLGKVVK QLRKHFGNNT IIRFANSSGG DLEVTTHSFN CGGEFFYCNT SGLFNSTWIS NTSVQGSNST GSNDSITLPC RIKQIINMWQ RIGQCMYAPP IQGVIRCVSN ITGLILTRDG GSTNSTTETF RPGGGDMRDN WRSELYKYKV VKIEPLGVAP TRAKRRVVGG GGGSGGGGSA VGIGAVRRGF LGAAGSTMGA ASMTLTVQAR NLLSGIVQQQ SNLLRAPEAQ QHLLKLTVWG IKQLQARVLA VERYLRDQQL LGIWGCSGKL ICTTNVPWNS SWSNRNLSEI WDNMTWLQWD KEISNYTQII YGLLEESQNQ QEKNEQDLLA LDGGGGSHHH HHHHH

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Macromolecule #2: Rabbit antibody E70 fragment antigen binding heavy chain

MacromoleculeName: Rabbit antibody E70 fragment antigen binding heavy chain
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QSLEESGGGL VKPGGTLTLT CKASGIDFTS GYDMCWVRQA PGKGLEWVAC IYLGDGNTYY ASWAKGQFTI SKTSSTTVTL QMTSLTAADT ATYFCARFAG YRYSVWSYPD LWGPGTLVTV SSGQPKAPSV FPLAPCCGDT PSSTVTLGCL VKGYLPEPVT VTWNSGTLTN ...String:
QSLEESGGGL VKPGGTLTLT CKASGIDFTS GYDMCWVRQA PGKGLEWVAC IYLGDGNTYY ASWAKGQFTI SKTSSTTVTL QMTSLTAADT ATYFCARFAG YRYSVWSYPD LWGPGTLVTV SSGQPKAPSV FPLAPCCGDT PSSTVTLGCL VKGYLPEPVT VTWNSGTLTN GVRTFPSVRQ SSGLYSLSSV VSVTSSSQPV TCNVAHPATN TKVDKTVAPS TCSK

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Macromolecule #3: Rabbit antibody E70 fragment antigen binding kappa chain

MacromoleculeName: Rabbit antibody E70 fragment antigen binding kappa chain
type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIVMTQTPAS VEADVGGTVT IKCQASQRIV NLVAWYQHKP GQPPKLLIIG ASDLASGVPS RFSGSGYGTE FTLTISDLEC ADAATYFCQS AYNGDGDNAF GGGTEVVVKG DPVAPSVLIF PPAADQVATG TVTIVCVANK YFPDVTVTWE VDGTTQTTGI ENSKTPQNSA ...String:
DIVMTQTPAS VEADVGGTVT IKCQASQRIV NLVAWYQHKP GQPPKLLIIG ASDLASGVPS RFSGSGYGTE FTLTISDLEC ADAATYFCQS AYNGDGDNAF GGGTEVVVKG DPVAPSVLIF PPAADQVATG TVTIVCVANK YFPDVTVTWE VDGTTQTTGI ENSKTPQNSA DCTYNLSSTL TLTSTQYNSH KEYTCKVTQG TTSVVQSFNR GDC

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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

Concentration0.03 mg/mL
BufferpH: 7.4
Component:
ConcentrationName
50.0 mMTris
150.0 mMSodium chloride
StainingType: NEGATIVE / Material: uranyl formate
Details: Grids were negatively stained with 2% (w/v) uranyl formate for 50 s
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 25.0 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4zmj


Details: Coordinates for ligand-free SOSIP trimer converted to EM map and low-pass filtered to 40 Angstrom
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 20865

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