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- EMDB-2285: 3D map of a peptide conjugated antibody particle by individual-pa... -

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Basic information

Entry
Database: EMDB / ID: EMD-2285
Title3D map of a peptide conjugated antibody particle by individual-particle electron tomography (IPET) and optimized negative-staining.
Map dataReconstruction of a peptide-conjugated human IgG1 antibody by optimized negative-staining and individual-particle electron tomography
Sample
  • Sample: Peptide-conjugated Human IgG1 Antibody
  • Protein or peptide: IgG AntibodyImmunoglobulin G
Keywordspeptide conjugated human IgG1 antibody
Biological speciesHomo sapiens (human)
Methodelectron tomography / negative staining / Resolution: 16.5 Å
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 /
Abstract: Peptides show much promise as potent and selective drug candidates. Fusing peptides to a scaffold monoclonal antibody produces a conjugated antibody which has the advantages of peptide activity yet ...Peptides show much promise as potent and selective drug candidates. Fusing peptides to a scaffold monoclonal antibody produces a conjugated antibody which has the advantages of peptide activity yet also has the pharmacokinetics determined by the scaffold antibody. However, the conjugated antibody often has poor binding affinity to antigens that may be related to unknown structural changes. The study of the conformational change is difficult by conventional techniques because structural fluctuation under equilibrium results in multiple structures co-existing. Here, we employed our two recently developed electron microscopy (EM) techniques: optimized negative-staining (OpNS) EM and individual-particle electron tomography (IPET). Two-dimensional (2D) image analyses and three-dimensional (3D) maps have shown that the domains of antibodies present an elongated peptide-conjugated conformational change, suggesting that our EM techniques may be novel tools to monitor the structural conformation changes in heterogeneous and dynamic macromolecules, such as drug delivery vehicles after pharmacological synthesis and development.
History
DepositionJan 18, 2013-
Header (metadata) releaseJan 30, 2013-
Map releaseJan 30, 2013-
UpdateFeb 13, 2013-
Current statusFeb 13, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2285.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a peptide-conjugated human IgG1 antibody by optimized negative-staining and individual-particle electron tomography
Voxel sizeX=Y=Z: 1.406 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-2.77701998 - 7.77161026
Average (Standard dev.)0.00285887 (±0.25459847)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-117-6
Dimensions200200200
Spacing200200200
CellA=B=C: 281.2 Å
α=β=γ: 90.0 °

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/NS7-11-6
NC/NR/NS200200200
D min/max/mean-2.7777.7720.003

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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
Components
  • Sample: Peptide-conjugated Human IgG1 Antibody
  • Protein or peptide: IgG AntibodyImmunoglobulin G

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Supramolecule #1000: Peptide-conjugated Human IgG1 Antibody

SupramoleculeName: Peptide-conjugated Human IgG1 Antibody / type: sample / ID: 1000
Details: The sample was thawed from storage at -80 degrees Celcius before being loaded onto the grid
Oligomeric state: Monomer / Number unique components: 1
Molecular weightExperimental: 160 KDa / Theoretical: 160 KDa

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Macromolecule #1: IgG Antibody

MacromoleculeName: IgG Antibody / type: protein_or_peptide / ID: 1 / Name.synonym: IgG / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Plasma
Molecular weightExperimental: 160 KDa / Theoretical: 160 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.4
Details: 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
StainingType: NEGATIVE
Details: EM 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 ...Details: EM 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.
GridDetails: 200 mesh glow-discharged thin carbon-coated EM grids (Cu-200CN, Pacific Grid-Tech, USA)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy #1

MicroscopeZEISS LIBRA120PLUS
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Gatan / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -70.5 ° / Tilt series - Axis1 - Max angle: 70.5 ° / Tilt series - Axis1 - Angle increment: 1.5 °
Microscopy ID1
Detailstilt step is 1.5 degree
DateMay 1, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 1.406 µm / Number real images: 95 / Average electron dose: 250 e/Å2 / Bits/pixel: 16

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Electron microscopy #2

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Gatan / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -70.5 ° / Tilt series - Axis1 - Max angle: 70.5 ° / Tilt series - Axis1 - Angle increment: 1.5 °
Microscopy ID2
Detailstilt step is 1.5 degree
DateOct 1, 2009
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 1.406 µm / Number real images: 95 / Average electron dose: 250 e/Å2 / Bits/pixel: 16

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Image processing

CTF correctionDetails: TOMOCTF
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.5 Å / Resolution method: OTHER
Software - Name: Individual-partcile, electron, tomogrphy, (IPET), and, FETR
Details: Map was reconstructed by individual-particle electron tomography (IPET)and Focus ET Reconstruction Algorithm.
Number images used: 95
DetailsReconstruction was done by Individual-Particle Tomography Reconstruction (IPET) method.{eulerAnglesDetails}: Tomography tilt angle from -70.5 to 70.5 in step of 1.5

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: Manual
DetailsManually 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.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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