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Yorodumi- EMDB-8268: HIV NFL trimer cross-linked with Glutaraldehyde and in complex wi... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8268 | |||||||||
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Title | HIV NFL trimer cross-linked with Glutaraldehyde and in complex with bnAb VRC01 | |||||||||
Map data | HIV NFL trimer cross-linked with Glutaraldehyde and in complex with bnAb VRC01 | |||||||||
Sample |
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Biological species | Human immunodeficiency virus 1 | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 20.0 Å | |||||||||
Authors | Ward AB / de Val N | |||||||||
Citation | Journal: PLoS Pathog / Year: 2016 Title: Thermostability of Well-Ordered HIV Spikes Correlates with the Elicitation of Autologous Tier 2 Neutralizing Antibodies. Authors: Yu Feng / Karen Tran / Shridhar Bale / Shailendra Kumar / Javier Guenaga / Richard Wilson / Natalia de Val / Heather Arendt / Joanne DeStefano / Andrew B Ward / Richard T Wyatt / Abstract: In the context of HIV vaccine design and development, HIV-1 spike mimetics displaying a range of stabilities were evaluated to determine whether more stable, well-ordered trimers would more ...In the context of HIV vaccine design and development, HIV-1 spike mimetics displaying a range of stabilities were evaluated to determine whether more stable, well-ordered trimers would more efficiently elicit neutralizing antibodies. To begin, in vitro analysis of trimers derived from the cysteine-stabilized SOSIP platform or the uncleaved, covalently linked NFL platform were evaluated. These native-like trimers, derived from HIV subtypes A, B, and C, displayed a range of thermostabilities, and were "stress-tested" at varying temperatures as a prelude to in vivo immunogenicity. Analysis was performed both in the absence and in the presence of two different adjuvants. Since partial trimer degradation was detected at 37°C before or after formulation with adjuvant, we sought to remedy such an undesirable outcome. Cross-linking (fixing) of the well-ordered trimers with glutaraldehyde increased overall thermostability, maintenance of well-ordered trimer integrity without or with adjuvant, and increased resistance to solid phase-associated trimer unfolding. Immunization of unfixed and fixed well-ordered trimers into animals revealed that the elicited tier 2 autologous neutralizing activity correlated with overall trimer thermostability, or melting temperature (Tm). Glutaraldehyde fixation also led to higher tier 2 autologous neutralization titers. These results link retention of trimer quaternary packing with elicitation of tier 2 autologous neutralizing activity, providing important insights for HIV-1 vaccine design. | |||||||||
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_8268.map.gz | 1.9 MB | EMDB map data format | |
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Header (meta data) | emd-8268-v30.xml emd-8268.xml | 11.4 KB 11.4 KB | Display Display | EMDB header |
Images | emd_8268.png | 16.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8268 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8268 | HTTPS FTP |
-Validation report
Summary document | emd_8268_validation.pdf.gz | 77.6 KB | Display | EMDB validaton report |
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Full document | emd_8268_full_validation.pdf.gz | 76.7 KB | Display | |
Data in XML | emd_8268_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8268 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8268 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_8268.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | HIV NFL trimer cross-linked with Glutaraldehyde and in complex with bnAb VRC01 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.1 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : BG505_NFL_trimer cross-linked with glutaraldehyde and in complex ...
Entire | Name: BG505_NFL_trimer cross-linked with glutaraldehyde and in complex with VRC01 bnAb |
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Components |
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-Supramolecule #1: BG505_NFL_trimer cross-linked with glutaraldehyde and in complex ...
Supramolecule | Name: BG505_NFL_trimer cross-linked with glutaraldehyde and in complex with VRC01 bnAb type: complex / ID: 1 / Parent: 0 Details: BG505_NFL_trimer cross-linked with glutaraldehyde and in complex with 3 broadly neutralizing antibodies VRC01 |
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Source (natural) | Organism: Human immunodeficiency virus 1 |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK 293F / Recombinant plasmid: pPPI4 |
Molecular weight | Theoretical: 570 KDa |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.01 mg/mL | |||||||||
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Buffer | pH: 7.4 Component:
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Staining | Type: NEGATIVE / Material: Uranyl Formate Details: Sample was stained using two applications of 3 uL 2% UF. | |||||||||
Grid | Model: Electron Microscopy Sciences / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Details: The grids were glow-discharged at 20 mA. | |||||||||
Details | The trimer was incubated at RT with a 10 M excess of the broadly neutralizing antibody VRC01. The complex was purified by SEC. |
-Electron microscopy
Microscope | FEI TECNAI SPIRIT |
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Image recording | Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number grids imaged: 1 / Number real images: 60 / Average electron dose: 36.0 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus min: 1.0 µm / Nominal magnification: 46000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
Experimental equipment | Model: Tecnai Spirit / Image courtesy: FEI Company |