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- EMDB-1970: Negative stained image reconstruction of HIV spike protein in com... -

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Basic information

Entry
Database: EMDB / ID: EMD-1970
TitleNegative stained image reconstruction of HIV spike protein in complex with PGT128 Fab at 14 Angstrom resolution
Map dataHIV Envelope protein in complex with Fab PGT128Env (gene)
Sample
  • Sample: HIV spike protein 664G construct in complex with Fab fragment of PGT128 monoclonal antibody
  • Protein or peptide: Human immunodeficiency virus envelope protein
  • Protein or peptide: PGT128 monoclonal antibody fragment
KeywordsHIV / broadly neutralizing antibody / glycan shield
Biological speciesHuman immunodeficiency virus / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 14.0 Å
AuthorsKhayat R / Pejchal R / Julien J-P / Wilson IA
CitationJournal: Science / Year: 2011
Title: A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.
Authors: Robert Pejchal / Katie J Doores / Laura M Walker / Reza Khayat / Po-Ssu Huang / Sheng-Kai Wang / Robyn L Stanfield / Jean-Philippe Julien / Alejandra Ramos / Max Crispin / Rafael Depetris / ...Authors: Robert Pejchal / Katie J Doores / Laura M Walker / Reza Khayat / Po-Ssu Huang / Sheng-Kai Wang / Robyn L Stanfield / Jean-Philippe Julien / Alejandra Ramos / Max Crispin / Rafael Depetris / Umesh Katpally / Andre Marozsan / Albert Cupo / Sebastien Maloveste / Yan Liu / Ryan McBride / Yukishige Ito / Rogier W Sanders / Cassandra Ogohara / James C Paulson / Ten Feizi / Christopher N Scanlan / Chi-Huey Wong / John P Moore / William C Olson / Andrew B Ward / Pascal Poignard / William R Schief / Dennis R Burton / Ian A Wilson /
Abstract: The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to ...The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man(9) at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificity. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.
History
DepositionSep 25, 2011-
Header (metadata) releaseOct 20, 2011-
Map releaseOct 21, 2011-
UpdateOct 21, 2011-
Current statusOct 21, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

1970.png

Downloads & links

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Map

FileDownload / File: emd_1970.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHIV Envelope protein in complex with Fab PGT128
Voxel sizeX=Y=Z: 2.18 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-1.63715 - 3.57025
Average (Standard dev.)0.0105322 (±0.257004)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 348.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.182.182.18
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z348.800348.800348.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-1.6373.5700.011

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Supplemental data

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Sample components

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Entire : HIV spike protein 664G construct in complex with Fab fragment of ...

EntireName: HIV spike protein 664G construct in complex with Fab fragment of PGT128 monoclonal antibody
Components
  • Sample: HIV spike protein 664G construct in complex with Fab fragment of PGT128 monoclonal antibody
  • Protein or peptide: Human immunodeficiency virus envelope protein
  • Protein or peptide: PGT128 monoclonal antibody fragment

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Supramolecule #1000: HIV spike protein 664G construct in complex with Fab fragment of ...

SupramoleculeName: HIV spike protein 664G construct in complex with Fab fragment of PGT128 monoclonal antibody
type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: One trimer binds to three Fabs / Number unique components: 2
Molecular weightExperimental: 445 KDa / Method: Size exclusion chromatography and light scattering

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Macromolecule #1: Human immunodeficiency virus envelope protein

MacromoleculeName: Human immunodeficiency virus envelope protein / type: protein_or_peptide / ID: 1 / Name.synonym: Spike protein
Details: The PGT128 Fab was complexed to the spike protein, partially deglycosylated, purified using size exclusion chromatography, and concentrated.
Number of copies: 3 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus / Strain: KNH1144 SOSIP 664G / synonym: Spike protien
Recombinant expressionOrganism: 293S / Recombinant plasmid: pP14

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Macromolecule #2: PGT128 monoclonal antibody fragment

MacromoleculeName: PGT128 monoclonal antibody fragment / type: protein_or_peptide / ID: 2 / Name.synonym: Fab fragment / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human

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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

Concentration0.1 mg/mL
BufferpH: 7.5 / Details: 20 mM HEPES pH 7.5, 50 mM NaCl
StainingType: NEGATIVE / Details: 2% Uranyl Formate for 30 seconds
GridDetails: 400 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.95 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 100000
Sample stageSpecimen holder: eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 55
TemperatureMin: 293 K / Max: 293 K / Average: 293 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Legacy - Electron beam tilt params: -2 mrad
DateAug 11, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 0.109 µm / Number real images: 357 / Average electron dose: 16 e/Å2 / Details: Data collected on CCD / Bits/pixel: 16
Tilt angle min0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Micrograph and each particle
Final two d classificationNumber classes: 62
Final angle assignmentDetails: SPIDER:theta 90 degrees, phi 120 degrees
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Sparx / Number images used: 12824
DetailsThe particles were selected using an DoG Picker, and cleaned using reference free class averaging.

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