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- EMDB-3120: cryoEM reconstruction of bnAb PGT128 in complex with BG505 SOSIP.... -

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Basic information

Entry
Database: EMDB / ID: EMD-3120
TitlecryoEM reconstruction of bnAb PGT128 in complex with BG505 SOSIP.664 Env trimer
Map dataReconstruction of PGT128 Fab in complex with BG505 SOSIP.664 Env trimer
Sample
  • Sample: PGT128 Fab bound to BG505 SOSIP.664 HIV-1 Env trimer
  • Protein or peptide: HIV-1 Envelope glycoprotein
  • Protein or peptide: Immunoglobulin G PGT128
KeywordsHIV-1 / Env / bnAb / antibody / PGT128
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / identical protein binding / membrane
Similarity search - Function
Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120
Similarity search - Domain/homology
Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.47 Å
AuthorsLee JH / de Val N / Lyumkis D / Ward AB
CitationJournal: Structure / Year: 2015
Title: Model Building and Refinement of a Natively Glycosylated HIV-1 Env Protein by High-Resolution Cryoelectron Microscopy.
Authors: Jeong Hyun Lee / Natalia de Val / Dmitry Lyumkis / Andrew B Ward /
Abstract: Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample ...Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle cryoelectron microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 Å resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.
History
DepositionAug 11, 2015-
Header (metadata) releaseSep 16, 2015-
Map releaseSep 30, 2015-
UpdateOct 21, 2015-
Current statusOct 21, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.032
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.032
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3120.png

Downloads & links

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Map

FileDownload / File: emd_3120.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of PGT128 Fab in complex with BG505 SOSIP.664 Env trimer
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: 0.032 / Movie #1: 0.032
Minimum - Maximum-0.06394063 - 0.13750669
Average (Standard dev.)0.00007542 (±0.0053754)
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-0.0640.1380.000

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Supplemental data

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Sample components

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Entire : PGT128 Fab bound to BG505 SOSIP.664 HIV-1 Env trimer

EntireName: PGT128 Fab bound to BG505 SOSIP.664 HIV-1 Env trimer
Components
  • Sample: PGT128 Fab bound to BG505 SOSIP.664 HIV-1 Env trimer
  • Protein or peptide: HIV-1 Envelope glycoprotein
  • Protein or peptide: Immunoglobulin G PGT128

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Supramolecule #1000: PGT128 Fab bound to BG505 SOSIP.664 HIV-1 Env trimer

SupramoleculeName: PGT128 Fab bound to BG505 SOSIP.664 HIV-1 Env trimer / type: sample / ID: 1000
Oligomeric state: Three monomers of PGT128 Fab bind one Env trimer
Number unique components: 2
Molecular weightTheoretical: 570 KDa

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Macromolecule #1: HIV-1 Envelope glycoprotein

MacromoleculeName: HIV-1 Envelope glycoprotein / type: protein_or_peptide / ID: 1 / Name.synonym: Env
Details: The Env sequence is from the clade A virus BG505, truncated at residue 664 of gp41, and contains stabilizing SOSIP mutations.
Number of copies: 3 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: BG505 / synonym: HIV-1
Molecular weightTheoretical: 420 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F

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Macromolecule #2: Immunoglobulin G PGT128

MacromoleculeName: Immunoglobulin G PGT128 / type: protein_or_peptide / ID: 2 / Name.synonym: IgG PGT128
Details: The fragment antigen binding (Fab) of PGT128 was used to form the complex.
Number of copies: 3 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 500 KDa
Recombinant expressionOrganism: Homo sapiens (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

Concentration2.5 mg/mL
BufferpH: 7.4 / Details: 50 mM Tris, 150 mM NaCl, 0.675 mM DDM
GridDetails: 400 mesh C-Flat CF-2/2-4C, plasma treated for 5 seconds
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 160 K / Instrument: HOMEMADE PLUNGER
Method: Grids were frozen using a manual plunger at 4 degrees.

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Objective astigmatism was corrected at 22,500x magnification
DateOct 7, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 2111 / Average electron dose: 33 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
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: 3.5 µm / Nominal defocus min: 1.5 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 was corrected at 22,500x magnification
DateNov 7, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 2111 / Average electron dose: 35 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
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: 3.5 µm / Nominal defocus min: 1.5 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

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.47 Å / Resolution method: OTHER / Software - Name: Imagic, Relion / Number images used: 92095

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