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- EMDB-3312: HIV-1 cleaved wild type JR-FL EnvdCT trimer in complex with PGT15... -

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Basic information

Entry
Database: EMDB / ID: EMD-3312
TitleHIV-1 cleaved wild type JR-FL EnvdCT trimer in complex with PGT151 and 10E8 Fabs at 8.8 A resolution
Map dataReconstruction of EnvdCT in complex with 10E8 and PGT151 Fabs. Partial density of a third 10E8 Fab visible due to partial binding occupancy.
Sample
  • Sample: Cleaved JR-FL EnvdCT in complex with PGT151 and 10E8 Fabs
  • Protein or peptide: HIV-1 Envelope glycoprotein
  • Protein or peptide: Immunoglobulin G PGT151
  • Protein or peptide: Immunoglobulin G 10E8
KeywordsHIV-1 / Env / PGT151 / 10E8 / antibody / MPER
Biological speciesHuman Immunodeficiency Virus-1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.8 Å
AuthorsLee JH / 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) releaseMar 9, 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: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3312.png

Downloads & links

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Map

FileDownload / File: emd_3312.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of EnvdCT in complex with 10E8 and PGT151 Fabs. Partial density of a third 10E8 Fab visible due to partial binding occupancy.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 256 pix.
= 335.36 Å		1.31 Å/pix.
x 256 pix.
= 335.36 Å		1.31 Å/pix.
x 256 pix.
= 335.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.01747882 - 0.05539626
Average (Standard dev.)0.00008552 (±0.00413677)
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-0.0170.0550.000

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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 and 10E8 Fabs

EntireName: Cleaved JR-FL EnvdCT in complex with PGT151 and 10E8 Fabs
Components
  • Sample: Cleaved JR-FL EnvdCT in complex with PGT151 and 10E8 Fabs
  • Protein or peptide: HIV-1 Envelope glycoprotein
  • Protein or peptide: Immunoglobulin G PGT151
  • Protein or peptide: Immunoglobulin G 10E8

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Supramolecule #1000: Cleaved JR-FL EnvdCT in complex with PGT151 and 10E8 Fabs

SupramoleculeName: Cleaved JR-FL EnvdCT in complex with PGT151 and 10E8 Fabs
type: sample / ID: 1000
Oligomeric state: One trimer bound to one PGT151 and two 10E8 Fabs
Number unique components: 3
Molecular weightTheoretical: 585 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: 1 / 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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Macromolecule #3: Immunoglobulin G 10E8

MacromoleculeName: Immunoglobulin G 10E8 / type: protein_or_peptide / ID: 3 / Name.synonym: IgG 10E8 / Details: 10E8 expressed as Fab / Number of copies: 2 / Oligomeric state: Heterodimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 50 MDa
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

Concentration4 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

Microscopy ID1
MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 22,500x magnification.
DateDec 16, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5.0 µm / Number real images: 3969 / 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. However, in the Nov session, the microscope experienced FEG instability resulting in ...Details: Each full dose image is an aligned stack of frames recorded each using a dose of ~10 e-/Angstrom^2/sec. However, in the Nov session, the microscope experienced FEG instability resulting in intensity drop over the course of data collection.
Tilt angle min0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #2

Microscopy ID2
MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 22,500x magnification.
DetailsUnstable beam intensity over the course of data collection.
DateNov 17, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5.0 µm / Number real images: 3969 / Average electron dose: 28.1 e/Å2
Details: Each full dose image is an aligned stack of frames recorded each using a dose of ~10 e-/Angstrom^2/sec. However, in the Nov session, the microscope experienced FEG instability resulting in ...Details: Each full dose image is an aligned stack of frames recorded each using a dose of ~10 e-/Angstrom^2/sec. However, in the Nov session, the microscope experienced FEG instability resulting in intensity drop over the course of data collection.
Tilt angle min0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe full data set was sorted into multiple 3D classes, which all had various Fab binding stoichiometries of PGT151 and 10E8. This reconstruction is the only sub-population that refined below 10A resolution.
CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.8 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 15525

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Atomic model buiding 1

Initial modelPDB ID:

4g6f


Chain - #0 - Chain ID: L / Chain - #1 - Chain ID: H / Chain - #2 - Chain ID: P
SoftwareName: Chimera
DetailsThe N-terminal helix of the gp41 peptide and Fab constant regions were removed prior to fitting.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

5fuu


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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