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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: 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
SamplePGT128 Fab bound to BG505 SOSIP.664 HIV-1 Env trimer:
HIV-1 Envelope glycoprotein / Immunoglobulin G PGT128
KeywordsHIV-1 / Env / bnAb / antibody / PGT128
SourceHuman immunodeficiency virus 1 (HIV-1) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 4.47 Å resolution
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.
DateDeposition: Aug 11, 2015 / Header (metadata) release: Sep 16, 2015 / Map release: Sep 30, 2015 / Last update: Oct 21, 2015

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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

Downloads & links

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Map

Fileemd_3120.map.gz (map file in CCP4 format, 65537 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.31 Å/pix.
= 335.36 Å
256 pix
1.31 Å/pix.
= 335.36 Å
256 pix
1.31 Å/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
Contour Level:0.032 (by author), 0.032 (movie #1):
Minimum - Maximum-0.06394063 - 0.13750669
Average (Standard dev.)0.00007542 (0.00537540)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin000
Limit255255255
Spacing256256256
CellA=B=C: 335.36 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
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 / Number of components: 2
Oligomeric State: Three monomers of PGT128 Fab bind one Env trimer
MassTheoretical: 570 kDa

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Component #1: protein, HIV-1 Envelope glycoprotein

ProteinName: HIV-1 Envelope glycoprotein / a.k.a: Env / Oligomeric Details: Trimer
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 / Recombinant expression: Yes
MassTheoretical: 420 kDa
SourceSpecies: Human immunodeficiency virus 1 (HIV-1) / Strain: BG505
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: HEK293F

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Component #2: protein, Immunoglobulin G PGT128

ProteinName: Immunoglobulin G PGT128 / a.k.a: IgG PGT128 / Oligomeric Details: Monomer
Details: The fragment antigen binding (Fab) of PGT128 was used to form the complex.
Recombinant expression: Yes / Number of Copies: 3
MassTheoretical: 500 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: HEK293F

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.5 mg/ml / Buffer solution: 50 mM Tris, 150 mM NaCl, 0.675 mM DDM / pH: 7.4
Support film400 mesh C-Flat CF-2/2-4C, plasma treated for 5 seconds
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 160 K / Humidity: 90 %
Method: Grids were frozen using a manual plunger at 4 degrees.

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Electron microscopy imaging #1

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Oct 7, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 33 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500 X (nominal), 22500 X (calibrated)
Astigmatism: Objective astigmatism was corrected at 22,500x magnification
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Electron microscopy imaging #2

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Nov 7, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 35 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500 X (nominal), 22500 X (calibrated)
Astigmatism: Objective astigmatism was corrected at 22,500x magnification
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2111 / Sampling size: 5 microns
Details: Each full dose image is an aligned stack of frames recorded each using a dose of ~10 e-/Angstrom^2/sec.

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C3 (3 fold cyclic) / Number of projections: 92095
3D reconstructionAlgorithm: Maximum likelihood / Software: Imagic, Relion / CTF correction: Each micrograph / Resolution: 4.47 Å / Resolution method: FSC 0.143, gold-standard

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