EMD-2285 Experiments and Validation

3D map of a peptide conjugated antibody particle by individual-particle electron tomography (IPET) and optimized negative-staining.

Tomographic reconstruction
Overview of EMD-2285
Sample name: Peptide-conjugated Human IgG1 Antibody
Organism: Homo sapiens
Related EM entry by publication: EMD-2286

Map parameters

Minimum density: -2.777
Maximum density: 7.772
Average density: 0.003
Standard deviation: 0.255
Recommended contour level: 1.00 (author)

Sample information

Sample name: Peptide-conjugated Human IgG1 Antibody
Protein: IgG Antibody

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: Individual-partcile, electron, tomogrphy, (IPET), and, FETR
Number of tilts (projections): 95
Tilt angle increments (degrees): 1.5
Reconstruction protocol: Focused ET reconstruction (FETR) algorithms
CTF correction: TOMOCTF
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.

Fitting

Initial atomic model: 1IGT
Fitting software: Manual
Fitting 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.

Imaging

Session 1
Microscope model: ZEISS LIBRA120PLUS
Electron source: LAB6
Electron dose (e/A**2): 250.0
Nominal CS (mm): 2.20
Nominal magnification: 80000.0
Defocus max (nm): 2,000.00
Defocus min (nm): 1,000.00
Tilt max (degrees): 70.50
Tilt min (degrees): -70.50
Holder model: OTHER
Details: tilt step is 1.5 degree
Session 2
Microscope model: FEI TECNAI 20
Electron source: LAB6
Electron dose (e/A**2): 250.0
Nominal CS (mm): 2.00
Nominal magnification: 80000.0
Defocus max (nm): 2,000.00
Defocus min (nm): 1,000.00
Tilt max (degrees): 70.50
Tilt min (degrees): -70.50
Holder model: OTHER
Details: tilt step is 1.5 degree
Acquisition
Number of images: 95
Sampling interval (μm): 1.4059999999999999

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.01
Buffer: 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
Staining procedure: 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.
Specimen support: 200 mesh glow-discharged thin carbon-coated EM grids (Cu-200CN, Pacific Grid-Tech, USA)
Vitrification
Apparatus: NONE
Cryogen: NONE