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- EMDB-3121: cryoEM reconstruction of bnAb PGT128 in complex with BG505 SOSIP.... -

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Basic information

Entry
Database: EMDB / ID: EMD-3121
TitlecryoEM reconstruction of bnAb PGT128 in complex with BG505 SOSIP.664 Env trimer at 4.4 Angstrom resolution
Map dataReconstruction of PGT128 Fab in complex with BG505 SOSIP.664 Env trimer at 4.4 Angstrom resolution.
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 / positive regulation of establishment of T cell polarity / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / apoptotic process / host cell plasma membrane / structural molecule activity / virion membrane / identical protein binding / plasma 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: 4.36 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: 0.041
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.041
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5aco
  • Surface level: 0.041
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3121.png

Downloads & links

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Map

FileDownload / File: emd_3121.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 at 4.4 Angstrom resolution.
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: 0.041 / Movie #1: 0.041
Minimum - Maximum-0.07047713 - 0.1443055
Average (Standard dev.)0.00008823 (±0.00531572)
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-0.0700.1440.000

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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: 4.36 Å / Resolution method: OTHER / Software - Name: Imagic, Relion
Details: At the end of refinement which gave the 4.47 Angstrom resolution structure, a mask excluding the Fab constant domain (flexible region) was applied, and refined for an additional 3 iterations.
Number images used: 92095

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Atomic model buiding 1

Initial modelPDB ID:

3tyg


Chain - #0 - Chain ID: H / Chain - #1 - Chain ID: L
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5aco:
Cryo-EM structure of PGT128 Fab in complex with BG505 SOSIP.664 Env trimer

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Atomic model buiding 2

Initial modelPDB ID:

4tvp


Chain - #0 - Chain ID: B / Chain - #1 - Chain ID: G
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5aco:
Cryo-EM structure of PGT128 Fab in complex with BG505 SOSIP.664 Env trimer

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