Cryo-EM Analysis of B1-scFv/B2-Fab Fusion

The samples B1-scFv/B2- Fab fusion and its complex with Ub-pY59 were negatively stained and observed on a Tecnai T12 microscope and a Tecnai T20 microscope (FEI Company) using the discharged continuous carbon grids as previously described,^{47 (link),48} respectively. Images were acquired at room temperature with a pixel size of 2.21 Å/pixel (T12, operated at 120 kV) or 3.319 Å/pixel (T20, operated at 200 kV) on the level of specimen using a 4K × 4K CCD camera (UltraScan 4000, Gatan Inc.). After all micrographs were visually screened, the contrast transfer function (CTF) was estimated for each micrograph by Gctf.^{49 (link)} The particle was selected using the Gautomatch (https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch) without template. Individual particles were extracted from the raw images with the 100 × 100 pixel window for T12 images or 80 × 80 pixel window for T20 images and were subjected to 25 cycles of 2D classification with a mask diameter of 200 Å for B1-scFv/B2-Fab or 220 Å for its complex with Ub-pY59 in Relion 3.0.^{50 (link)} The output 2D averages after several rounds were analyzed by comparing with the available protein structures utilized in this design.

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Zhou X.X., Bracken C.J., Zhang K., Zhou J., Mou Y., Wang L., Cheng Y., Leung K.K, & Wells J.A. (2020). Targeting Phosphotyrosine in Native Proteins with Conditional, Bispecific Antibody Traps. Journal of the American Chemical Society, 142(41), 17703-17713.

Publication 2020

Tecnai T12 microscope Tecnai T20 microscope UltraScan 4000

Acquired temperature Carbon Microscope Protein

Corresponding Organization : University of California, San Francisco

Other organizations : Howard Hughes Medical Institute

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Variable analysis

independent variables

Microscopes used: Tecnai T12 and Tecnai T20

dependent variables

Observations of the samples B1-scFv/B2-Fab fusion and its complex with Ub-pY59

control variables

Discharged continuous carbon grids used for sample preparation
Pixel size of 2.21 Å/pixel (T12) or 3.319 Å/pixel (T20)
Contrast transfer function (CTF) estimation using Gctf
Particle selection using Gautomatch without template
2D classification in Relion 3.0 with specific mask diameters

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