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- EMDB-3309: HIV-1 cleaved wild type JR-FL EnvdCT trimer in complex with PGT15... -

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Basic information

Entry
Database: EMDB / ID: EMD-3309
TitleHIV-1 cleaved wild type JR-FL EnvdCT trimer in complex with PGT151 at 4.3A resolution
Map dataReconstruction of wild type JR-FL EnvdCT-PGT151 Fab complex.
Sample
  • Sample: Cleaved JR-FL EnvdCT in complex with PGT151 Fab
  • Protein or peptide: HIV-1 Envelope glycoprotein
  • Protein or peptide: Immunoglobulin G PGT151
KeywordsHIV-1 / Env / PGT151 / broadly neutralizing antibody
Biological speciesHuman Immunodeficiency Virus-1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsLee JH / Ozorowski G / Ward AB
CitationJournal: Science / Year: 2016
Title: Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer.
Authors: Jeong Hyun Lee / Gabriel Ozorowski / Andrew B Ward /
Abstract: The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4(+) T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making ...The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4(+) T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making it difficult to study in its native state. Soluble stabilized trimers have provided valuable insights into the Env structure, but they lack the hydrophobic membrane proximal external region (MPER, an important target of broadly neutralizing antibodies), the transmembrane domain, and the cytoplasmic tail. Here we present (i) a cryogenic electron microscopy (cryo-EM) structure of a clade B virus Env, which lacks only the cytoplasmic tail and is stabilized by the broadly neutralizing antibody PGT151, at a resolution of 4.2 angstroms and (ii) a reconstruction of this form of Env in complex with PGT151 and MPER-targeting antibody 10E8 at a resolution of 8.8 angstroms. These structures provide new insights into the wild-type Env structure.
History
DepositionJan 29, 2016-
Header (metadata) releaseFeb 10, 2016-
Map releaseMar 9, 2016-
UpdateMar 9, 2016-
Current statusMar 9, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3309.png

Downloads & links

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Map

FileDownload / File: emd_3309.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of wild type JR-FL EnvdCT-PGT151 Fab complex.
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.04313641 - 0.13069391
Average (Standard dev.)0.00011561 (±0.00519857)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z335.360335.360335.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0430.1310.000

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Supplemental data

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Sample components

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Entire : Cleaved JR-FL EnvdCT in complex with PGT151 Fab

EntireName: Cleaved JR-FL EnvdCT in complex with PGT151 Fab
Components
  • Sample: Cleaved JR-FL EnvdCT in complex with PGT151 Fab
  • Protein or peptide: HIV-1 Envelope glycoprotein
  • Protein or peptide: Immunoglobulin G PGT151

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Supramolecule #1000: Cleaved JR-FL EnvdCT in complex with PGT151 Fab

SupramoleculeName: Cleaved JR-FL EnvdCT in complex with PGT151 Fab / type: sample / ID: 1000 / Oligomeric state: Two PGT151 Fabs bind one Env trimer / Number unique components: 2
Molecular weightTheoretical: 535 KDa

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Macromolecule #1: HIV-1 Envelope glycoprotein

MacromoleculeName: HIV-1 Envelope glycoprotein / type: protein_or_peptide / ID: 1 / Name.synonym: HIV-1 Env
Details: wild-type JR-FL Env trimer with the cytoplasmic tail truncated
Number of copies: 1 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human Immunodeficiency Virus-1 / Strain: JR-FL / synonym: HIV-1
Molecular weightTheoretical: 435 KDa
Recombinant expressionOrganism: Mammalian (mammals) / Recombinant strain: Human / Recombinant cell: HEK293F / Recombinant plasmid: phCMV3

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Macromolecule #2: Immunoglobulin G PGT151

MacromoleculeName: Immunoglobulin G PGT151 / type: protein_or_peptide / ID: 2 / Name.synonym: IgG PGT151 / Details: PGT151 cleaved into Fab / Number of copies: 2 / Oligomeric state: Heterodimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 50 KDa
Recombinant expressionOrganism: Mammalian (mammals) / Recombinant strain: Human / Recombinant cell: HEK293F

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.5 mg/mL
BufferpH: 7.4
Details: 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% DDM, 0.03 mg/mL sodium deoxycholate
GridDetails: 400 mesh C-Flat, CF-2/2-4C, plasma cleaned for 5 seconds
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Samples were treated with biobeads prior to freezing
Method: Grids were manually plunged at RT.

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Electron microscopy #1

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Microscopy ID1
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 22,500x magnification.
DateFeb 27, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5.0 µm / Number real images: 8759 / Average electron dose: 32.4 e/Å2
Details: Each full dose image is an aligned stack of frames recorded each using a dose of ~10 e-/Angstrom^2/sec.
Tilt angle min0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #2

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Microscopy ID2
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 22,500x magnification.
DateNov 8, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5.0 µm / Number real images: 8759 / Average electron dose: 35.1 e/Å2
Details: Each full dose image is an aligned stack of frames recorded each using a dose of ~10 e-/Angstrom^2/sec.
Tilt angle min0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #3

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Microscopy ID3
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 22,500x magnification.
DateOct 24, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5.0 µm / Number real images: 8759 / Average electron dose: 33.7 e/Å2
Details: Each full dose image is an aligned stack of frames recorded each using a dose of ~10 e-/Angstrom^2/sec.
Tilt angle min0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #4

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Microscopy ID4
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 22,500x magnification.
DateSep 11, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5.0 µm / Number real images: 8759 / Average electron dose: 32.2 e/Å2
Details: Each full dose image is an aligned stack of frames recorded each using a dose of ~10 e-/Angstrom^2/sec.
Tilt angle min0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 201386

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Atomic model buiding 1

Initial modelPDB ID:

4tvp


Chain - #0 - Chain ID: B / Chain - #1 - Chain ID: G
SoftwareName: Chimera
DetailsThe trimer was used as an initial model for model building and refinement.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

4nug


Chain - #0 - Chain ID: H / Chain - #1 - Chain ID: L
SoftwareName: Chimera
DetailsThe Fab was used as an initial model for model building and refinement.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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