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- EMDB-2286: 3D map of another peptide conjugated antibody particle by individ... -

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Basic information

Entry
Database: EMDB / ID: 2286
Title3D map of another peptide conjugated antibody particle by individual-particle electron tomography (IPET) and optimized negative-staining.
Map dataReconstruction of another IgG1 antibody by Individual-Particle Electron Microscopy (IPET).
SamplePeptide-conjugated Human IgG1 Antibody:
IgG AntibodyImmunoglobulin G
Keywordspeptide conjugated human IgG1 antibody
SourceHomo sapiens (human)
Methodelectron tomography / negative staining / 16.6 Å resolution
AuthorsTong H / Zhang L / Kaspar A / Rames M / Huang L / Woodnutt G / Ren G
CitationJournal: Sci Rep / Year: 2013
Title: Peptide-conjugation induced conformational changes in human IgG1 observed by optimized negative-staining and individual-particle electron tomography.
Authors: Huimin Tong / Lei Zhang / Allan Kaspar / Matthew J Rames / Liqing Huang / Gary Woodnutt / Gang Ren
DateDeposition: Jan 18, 2013 / Header (metadata) release: Jan 30, 2013 / Map release: Jan 30, 2013 / Last update: Feb 6, 2013

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.9
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.9
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_2286.map.gz (map file in CCP4 format, 31251 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
200 pix
1.41 Å/pix.
= 281.2 Å
200 pix
1.41 Å/pix.
= 281.2 Å
200 pix
1.41 Å/pix.
= 281.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.406 Å
Density
Contour Level:0.9 (by author), 0.9 (movie #1):
Minimum - Maximum-4.67092180 - 6.32597065
Average (Standard dev.)0.00124828 (0.24766813)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions200200200
Origin-6-3-4
Limit193196195
Spacing200200200
CellA=B=C: 281.2 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.4061.4061.406
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z281.200281.200281.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-3-6-4
NC/NR/NS200200200
D min/max/mean-4.6716.3260.001

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Supplemental data

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Sample components

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Entire Peptide-conjugated Human IgG1 Antibody

EntireName: Peptide-conjugated Human IgG1 Antibody
Details: The sample was thawed from storage at -80 degrees Celcius before being loaded onto the grid
Number of components: 1 / Oligomeric State: Monomer
MassTheoretical: 160 kDa / Experimental: 160 kDa

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Component #1: protein, IgG Antibody

ProteinName: IgG AntibodyImmunoglobulin G / a.k.a: IgG / Oligomeric Details: Monomer / Recombinant expression: No / Number of Copies: 1
MassTheoretical: 160 kDa / Experimental: 160 kDa
SourceSpecies: Homo sapiens (human)
Source (natural)Location in cell: Plasma

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionSpecimen conc.: 0.01 mg/ml
Buffer solution: 1X Dulbeccos phosphate-buffered saline (Invitrogen, La Jolla, CA), 2.7 mM KCl, 1.46 mM KH2PO4, 136.9 mM NaCl, and 8.1 mM Na2HPO4
pH: 7.4
Support film200 mesh glow-discharged thin carbon-coated EM grids (Cu-200CN, Pacific Grid-Tech, USA)
StainingEM Specimens were prepared by optimized negative-staining EM specimen preparation protocol as described Zhang L. and Ren G, Journal of Lipid Research, (2010) 51, 1228-1236; (2011) 52, 175-84 and BBA (2013) 1830, 2150-9. In brief, antibody was diluted to 0.01 mg/ml with deionized water. Aliquots (about 3ul) were applied to the 200 mesh glow-discharged thin carbon-coated EM grids (Cu-200CN, Pacific Grid-Tech, USA). The grid was washed by deionized water for three times, and then washed by 1% uranyl formate for three times before blotting to drying.
VitrificationInstrument: NONE / Cryogen name: NONE

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Electron microscopy imaging #1

ImagingMicroscope: ZEISS LIBRA120PLUS / Date: May 1, 2012 / Details: tilt step is 1.5 degree
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 250 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 80000 X (nominal) / Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 2000 nm
Specimen HolderHolder: Gatan / Model: OTHER / Tilt Angle: -70.5 - 70.5 deg.
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Electron microscopy imaging #2

ImagingMicroscope: FEI TECNAI 20 / Date: Oct 1, 2009 / Details: tilt step is 1.5 degree
Electron gunElectron source: LAB6 / Accelerating voltage: 200 kV / Electron dose: 250 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 80000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 2000 nm
Specimen HolderHolder: Gatan / Model: OTHER / Tilt Angle: -70.5 - 70.5 deg.
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 95 / Sampling size: 1.406 microns / Bit depth: 16

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Image processing

ProcessingMethod: electron tomography / Applied symmetry: C1 (asymmetric) / Tilt angle increment: 1.5 deg. / Number of sections: 95
Details: Reconstruction was done by Individual-Particle Tomography Reconstruction (IPET) method.
3D reconstructionAlgorithm: Focused ET reconstruction (FETR) algorithms
Euler angles: Tomography tilt angle from -70.5 to 70.5 in step of 1.5
Software: Individual-partcile, electron, tomogrphy, (IPET), and, FETR
CTF correction: TOMOCTF
Details: Map was reconstructed by individual-particle electron tomography (IPET)and Focus ET Reconstruction Algorithm.
Resolution: 16.6 Å / Resolution method: Intra-FSC.

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Atomic model buiding

Modeling #1Software: Manual / Refinement protocol: rigid body / Refinement space: REAL
Details: Manually fit each domain into the density map in order to compare the conformational different between the peptide-conjugated antibody structure and PDB structure in term of domain size and shape.
Input PDB model: 1IGT

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