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Title:3D map of a peptide conjugated antibody particle by individual-particle electron tomography (IPET) and optimized negative-staining.
Authors:Tong H, Zhang L, Kaspar A, Rames M, Huang L, Woodnutt G, Ren G
Sample:Peptide-conjugated Human IgG1 Antibody
Method:Tomography (16.5 angstroms resolution)
Red flagLatest update:2013-02-13
Sample
Sample name: Peptide-conjugated Human IgG1 Antibody
Oligomeric state: Monomer
Experimental molecular weight of the sample: 0.16
Components:
ID Type Name Exp. MW (MDa) Theo. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinIgG Antibody0.160.16MonomerfalseHomo sapiens
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.40.01 mg/mL1X Dulbeccos phosphate-buffered saline (Invitrogen, La Jolla, CA), 2.7 mM KCl, 1.46 mM KH2PO4, 136.9 mM NaCl, and 8.1 mM Na2HPO4EM 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.200 mesh glow-discharged thin carbon-coated EM grids (Cu-200CN, Pacific Grid-Tech, USA)
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
NONE% KNONE ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
ZEISS LIBRA120PLUS120 kVFLOOD BEAMBRIGHT FIELD2.2 mm1000 nm2000 nm80000LAB6GATAN ULTRASCAN 4000 (4k x 4k) mm
FEI TECNAI 20200 kVFLOOD BEAMBRIGHT FIELD2.0 mm1000 nm2000 nm80000LAB6GATAN ULTRASCAN 4000 (4k x 4k) mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
GatanOTHER-70.5°70.5° eV K K K mrad250 e/Å2tilt step is 1.5 degree01-MAY-2012
GatanOTHER-70.5°70.5° eV K K K mrad250 e/Å2tilt step is 1.5 degree01-OCT-2009
Processing
Protocol:Focused ET reconstruction (FETR) algorithms
Software:Individual-partcile, electron, tomogrphy, (IPET), and, FETR
CTF correction:TOMOCTF
Number of tilts:95
Tilt angle increment:1.5°
Imposed symmetry:C1
Resolution by author:16.5 Å
Resolution method:Intra-FSC.
Processing details:Map was reconstructed by individual-particle electron tomography (IPET)and Focus ET Reconstruction Algorithm. Reconstruction was done by Individual-Particle Tomography Reconstruction (IPET) method.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
951.406 μm/pixel16
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
1IGT rigid bodyManualREALManually 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.