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Yorodumi- EMDB-20274: Negative stain reconstruction of HIV-1 Env 16055 NFL TD 2CC+ trim... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-20274 | |||||||||||||||
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Title | Negative stain reconstruction of HIV-1 Env 16055 NFL TD 2CC+ trimer in complex with rabbit antibody 1C2 fragment antigen binding | |||||||||||||||
Map data | negative-stain 3D reconstruction | |||||||||||||||
Sample |
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Biological species | Human immunodeficiency virus 1 / Oryctolagus cuniculus (rabbit) | |||||||||||||||
Method | single particle reconstruction / negative staining / Resolution: 21.0 Å | |||||||||||||||
Authors | Torres JL / Ozorowski G / Andrew AB | |||||||||||||||
Funding support | United States, 4 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_20274.map.gz | 11.9 MB | EMDB map data format | |
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Header (meta data) | emd-20274-v30.xml emd-20274.xml | 20.8 KB 20.8 KB | Display Display | EMDB header |
Images | emd_20274.png | 70.1 KB | ||
Others | emd_20274_half_map_1.map.gz emd_20274_half_map_2.map.gz | 11.9 MB 11.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-20274 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-20274 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_20274.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | negative-stain 3D reconstruction | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.05 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: half map 1
File | emd_20274_half_map_1.map | ||||||||||||
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Annotation | half map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map 2
File | emd_20274_half_map_2.map | ||||||||||||
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Annotation | half map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : HIV-1 Env 16055 NFL TD 2CC+ trimer in complex with rabbit antibod...
Entire | Name: HIV-1 Env 16055 NFL TD 2CC+ trimer in complex with rabbit antibody 1C2 fragment antigen binding |
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Components |
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-Supramolecule #1: HIV-1 Env 16055 NFL TD 2CC+ trimer in complex with rabbit antibod...
Supramolecule | Name: HIV-1 Env 16055 NFL TD 2CC+ trimer in complex with rabbit antibody 1C2 fragment antigen binding type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Molecular weight | Theoretical: 570 KDa |
-Macromolecule #1: HIV-1 Env 16055 NFL TD 2CC+ trimer
Macromolecule | Name: HIV-1 Env 16055 NFL TD 2CC+ trimer / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Human immunodeficiency virus 1 |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MPMGSLQPLA TLYLLGMLVA SVLANGNLWV TVYYGVPVWK DAETTLFCAS DAKAYEKEKH NVWATHACVP TDPNPQEMVL ENVTENFNMW KNDMVEQMHT DVISLWDQSL KPCVKLTPLC VTLECRQVNT TNATSSVNVT NGEEIKNCSF NATTELRDKK QKVYALFYRL ...String: MPMGSLQPLA TLYLLGMLVA SVLANGNLWV TVYYGVPVWK DAETTLFCAS DAKAYEKEKH NVWATHACVP TDPNPQEMVL ENVTENFNMW KNDMVEQMHT DVISLWDQSL KPCVKLTPLC VTLECRQVNT TNATSSVNVT NGEEIKNCSF NATTELRDKK QKVYALFYRL DIVPLEEERK GNSSKYRLIN CNTSACTQAC PKVTFDPIPI HYCAPAGYAI LKCNNKTFNG TGPCNNVSTV QCTHGIKPVV STQLLLNGSL AEGEIIIRSE NLTNNVKTII VHLNESVEIV CTRPNNYTRK SIRIGPGQTF YATGDIIGNI RQAYCNISKD DWIRTLQRVG KKLAEHFPRR IINFTSPAGG DLEITTHSFN CRGEFFYCNT SSLFNSTYNP NDTNSNSSSS NSSLDITIPC RIKQIINMWQ RVGQCMYAPP IEGNITCKSN ITGLLLVRDG GVESNETEIF RPGGGDMRNN WRSELYKYKV VEIKPLGIAP TRCKRRVVEG GGGSGGGGSD DDDKAVGLGA VRRGFLGAAG STMGAASITL TVQARQLLSG IVQQQSNLLK APEAQQHLLQ LGVWGIKQLQ TRVLAIERYL KDQQLLGIWG CSGKLICTTA VPWNSSWSNK SHDEIWGNMT WMQWDREIGN YTNTIYRLLE DSQNQQEQNE KDLLACDGGG GSHHHHHHHH |
-Macromolecule #2: Rabbit antibody 1C2 fragment antigen binding heavy chain
Macromolecule | Name: Rabbit antibody 1C2 fragment antigen binding heavy chain type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MYRMQLLSCI ALSLALVTNS QCQSLEESGG DLVKPGASLT LTCTASGFSF GWNDYMSWVR QAPGKGLEWI GCIYAGSTRS TYYANWAKGR LTISKTSSTA VTLQMTSLTA ADTATYFCAR GAVTYDGLGG AYLKHFNLWG PGTLVTVSSG QPKAPSVFPL APCCGDTPSS ...String: MYRMQLLSCI ALSLALVTNS QCQSLEESGG DLVKPGASLT LTCTASGFSF GWNDYMSWVR QAPGKGLEWI GCIYAGSTRS TYYANWAKGR LTISKTSSTA VTLQMTSLTA ADTATYFCAR GAVTYDGLGG AYLKHFNLWG PGTLVTVSSG QPKAPSVFPL APCCGDTPSS TVTLGCLVKG YLPEPVTVTW NSGTLTNGVR TFPSVRQSSG LYSLSSVVSV TSSSQPVTCN VAHPATNTKV DKTVAPSTCS KHHHHHHHH |
-Macromolecule #3: Rabbit antibody 1C2 fragment antigen binding kappa chain
Macromolecule | Name: Rabbit antibody 1C2 fragment antigen binding kappa chain type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MYRMQLLSCI ALSLALVTNS AIKMTQTPSS VSAAVGGTVT VNCRASEDIE SYLAWYQQKP GQPPKLLIYD TSKLASGVPS RFKGSGSGTQ FALTISGVQC DDAATYYCLY GYISSDRIDF GFGGGTELVV KGDPVAPSVL IFPPAADQVA TGTVTIVCVA NKYFPDVTVT ...String: MYRMQLLSCI ALSLALVTNS AIKMTQTPSS VSAAVGGTVT VNCRASEDIE SYLAWYQQKP GQPPKLLIYD TSKLASGVPS RFKGSGSGTQ FALTISGVQC DDAATYYCLY GYISSDRIDF GFGGGTELVV KGDPVAPSVL IFPPAADQVA TGTVTIVCVA NKYFPDVTVT WEVDGTTQTT GIENSKTPQN SADCTYNLSS TLTLTSTQYN SHKEYTCKVT QGTTSVVQSF NRGDC |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.03 mg/mL | ||||||
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Buffer | pH: 7.4 Component:
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Staining | Type: NEGATIVE / Material: uranyl formate Details: Grids were negatively stained with 2% (w/v) uranyl formate for 50 s |
-Electron microscopy
Microscope | FEI TECNAI SPIRIT |
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Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Illumination 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 stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
Image recording | Film 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 |
-Image processing
CTF correction | Software - Name: Gctf (ver. 1.06) |
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Startup model | Type of model: PDB ENTRY PDB model - PDB ID: Details: Coordinates for ligand-free SOSIP trimer converted to EM map and low-pass filtered to 40 Angstrom |
Initial angle assignment | Type: OTHER / Software - Name: RELION (ver. 3.0) |
Final 3D classification | Software - Name: RELION (ver. 3.0) |
Final angle assignment | Type: OTHER / Software - Name: RELION (ver. 3.0) |
Final reconstruction | Applied 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: 6000 |