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Bis-(t-Boc)-bovine-insulin (BBI) was prepared as described [R. Geiger, H. H. Schöne, W. Pfaff, Physiol. Chem. 352, 1487 (1971)]. BBI (1 mg) and mono-NHS-Nanogold (30 nmol) (Nanoprobes, Stoneybrook, NY) were dissolved in 200 μl of N′N-dimethylacetamide containing 2 μl of di-isopropylethylamine, pH 7.6. After vigorous mixing at room temperature for 60 min the mixture was vacuum dried. The pellet obtained was dissolved in 20 μl of trifluoroacetic acid, kept at room temperature for 5 min, and then dried again. The pellet, resuspended in 120 μl of 1 M acetic acid, was chromatographed twice on a BioGel P10 column (1.7 cm by 25 cm) in 1 M acetic acid. The NGBI, >95% pure, had a molecular mass of 19,796 daltons by matrix-assisted laser desorption/ ionization (MALDI) time-of-flight (TOF) mass spectrometry (Fig. 1, inset), consistent with one insulin per Nanogold cluster.
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0344928877
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note
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-6 M) at 4°C overnight in 20 mM Hepes buffer (pH 7.5). Unbound NGBI was removed by microfiltration with a size cut-off of 300 kD (Sigma). The mixture was diluted to 7.5 μg of receptor protein per milliliter in 20 mM Hepes buffer (pH 7.5) for STEM.
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13
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0344928876
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note
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The specimen (5 μl) was injected into 5 μl of the Hepes buffer on a grid overlaid with a 23 Å carbon film on fenestrated celluloseacetobutyrate, then washed with Hepes buffer and 10 mM ammonium acetate (pH 7.5). The grid was wicked with filter paper, leaving a very thin solution layer, then quick-frozen in liquid ethane at -150°C. The frozen specimen was transferred at below -140°C into the STEM (Vacuum Generators, Model HB601UX) cold-stage and freeze-dried at -140°C. We acquired simultaneous elastic and inelastic digital images with 6.5 Å pixels using single-electron counting at 100 kV, with the specimen at - 150°C. The beam size was 3 Å.
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14
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0039201806
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Plenum, New York, ed. 2
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Paired elastic and inelastic images were combined for a fourfold signal increase over the elastic signal alone for carbonaceous specimens [R. F. Egerton, Electron Energy-Loss Spectroscopy in the electron Microscope (Plenum, New York, ed. 2, 1996), p. 145]. Single particles were interactively selected and low-pass filtered to 1.0 nm with a Gaussian filter with the programs WEB and SPIDER (Wadsworth Laboratories, Albany, NY). The molecular mass relative to the 23 Å carbon support had a Gaussian distribution with a modal mass of 570 kD, including 480 kD for the IR-NGBI plus ∼150 Triton X100 molecules. We computed orientations and 3D reconstructions as before [N. A. Farrow and F. P. Ottensmeyer, J. Opt. Soc. Am. A9, 1749 (1992); Ultramicroscopy 52, 141 (1993) ] on an SGI Indigo workstation (Silicon Graphics, Mountain View, CA) using filtered back-projection and an angular distribution-dependent filter. Resolution measurements were obtained by means of Fourier shell-phase residual calculations between reconstructions from two independent sets of half of the 704 images [G. J. Czarnota, D. W. Andrews, N. A. Farrow, F. P. Ottensmeyer, J. Struct. Biol. 113, 35 (1994)]. INSIGHT II (Molecular Simulations, San Diego, CA) served to insert known crystal structures and approximate models. Handedness was determined by fitting the crystallographic structure of TK into mirror pairs of the reconstruction.
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Electron Energy-loss Spectroscopy in the Electron Microscope
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Egerton, R.F.1
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Paired elastic and inelastic images were combined for a fourfold signal increase over the elastic signal alone for carbonaceous specimens [R. F. Egerton, Electron Energy-Loss Spectroscopy in the electron Microscope (Plenum, New York, ed. 2, 1996), p. 145]. Single particles were interactively selected and low-pass filtered to 1.0 nm with a Gaussian filter with the programs WEB and SPIDER (Wadsworth Laboratories, Albany, NY). The molecular mass relative to the 23 Å carbon support had a Gaussian distribution with a modal mass of 570 kD, including 480 kD for the IR-NGBI plus ∼150 Triton X100 molecules. We computed orientations and 3D reconstructions as before [N. A. Farrow and F. P. Ottensmeyer, J. Opt. Soc. Am. A9, 1749 (1992); Ultramicroscopy 52, 141 (1993) ] on an SGI Indigo workstation (Silicon Graphics, Mountain View, CA) using filtered back-projection and an angular distribution-dependent filter. Resolution measurements were obtained by means of Fourier shell-phase residual calculations between reconstructions from two independent sets of half of the 704 images [G. J. Czarnota, D. W. Andrews, N. A. Farrow, F. P. Ottensmeyer, J. Struct. Biol. 113, 35 (1994)]. INSIGHT II (Molecular Simulations, San Diego, CA) served to insert known crystal structures and approximate models. Handedness was determined by fitting the crystallographic structure of TK into mirror pairs of the reconstruction.
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Ottensmeyer, F.P.2
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Paired elastic and inelastic images were combined for a fourfold signal increase over the elastic signal alone for carbonaceous specimens [R. F. Egerton, Electron Energy-Loss Spectroscopy in the electron Microscope (Plenum, New York, ed. 2, 1996), p. 145]. Single particles were interactively selected and low-pass filtered to 1.0 nm with a Gaussian filter with the programs WEB and SPIDER (Wadsworth Laboratories, Albany, NY). The molecular mass relative to the 23 Å carbon support had a Gaussian distribution with a modal mass of 570 kD, including 480 kD for the IR-NGBI plus ∼150 Triton X100 molecules. We computed orientations and 3D reconstructions as before [N. A. Farrow and F. P. Ottensmeyer, J. Opt. Soc. Am. A9, 1749 (1992); Ultramicroscopy 52, 141 (1993) ] on an SGI Indigo workstation (Silicon Graphics, Mountain View, CA) using filtered back-projection and an angular distribution-dependent filter. Resolution measurements were obtained by means of Fourier shell-phase residual calculations between reconstructions from two independent sets of half of the 704 images [G. J. Czarnota, D. W. Andrews, N. A. Farrow, F. P. Ottensmeyer, J. Struct. Biol. 113, 35 (1994)]. INSIGHT II (Molecular Simulations, San Diego, CA) served to insert known crystal structures and approximate models. Handedness was determined by fitting the crystallographic structure of TK into mirror pairs of the reconstruction.
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Paired elastic and inelastic images were combined for a fourfold signal increase over the elastic signal alone for carbonaceous specimens [R. F. Egerton, Electron Energy-Loss Spectroscopy in the electron Microscope (Plenum, New York, ed. 2, 1996), p. 145]. Single particles were interactively selected and low-pass filtered to 1.0 nm with a Gaussian filter with the programs WEB and SPIDER (Wadsworth Laboratories, Albany, NY). The molecular mass relative to the 23 Å carbon support had a Gaussian distribution with a modal mass of 570 kD, including 480 kD for the IR-NGBI plus ∼150 Triton X100 molecules. We computed orientations and 3D reconstructions as before [N. A. Farrow and F. P. Ottensmeyer, J. Opt. Soc. Am. A9, 1749 (1992); Ultramicroscopy 52, 141 (1993) ] on an SGI Indigo workstation (Silicon Graphics, Mountain View, CA) using filtered back-projection and an angular distribution-dependent filter. Resolution measurements were obtained by means of Fourier shell-phase residual calculations between reconstructions from two independent sets of half of the 704 images [G. J. Czarnota, D. W. Andrews, N. A. Farrow, F. P. Ottensmeyer, J. Struct. Biol. 113, 35 (1994)]. INSIGHT II (Molecular Simulations, San Diego, CA) served to insert known crystal structures and approximate models. Handedness was determined by fitting the crystallographic structure of TK into mirror pairs of the reconstruction.
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We thank Y. Mao, M. Burke, B. Rutherford, and H. Hsu for their technical assistance. Supported by grants from the Medical Research Council of Canada, the National Cancer Institute of Canada, and by funds from Cancer Care Ontario.
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