Antibody-Based Affinity Cryoelectron Microscopy at 2.6-Å Resolution

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Antibody-Based Affinity Cryoelectron Microscopy at 2.6-Å Resolution Guimei Yu, Kunpeng Li, Pengwei Huang, Xi Jiang, Wen Jiang Structure Volume 24, Issue 11, Pages 1984-1990 (November 2016) DOI: 10.1016/j.str.2016.09.008 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 1984-1990DOI: (10.1016/j.str.2016.09.008) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Antibody-Based Affinity Cryo-EM of TV (A) Schematic diagram of TV immobilized on the antibody-based affinity grid. For clear visualization it was not drawn to scale. (B and C) Affinity cryo-EM micrographs of TV frozen using the optimized condition at 500× (B) and 3,800× (C) magnifications. Local ice thicknesses were estimated using the Beer-Lambert law-based method. (D) A representative motion-corrected micrograph. (E) The 2D power spectrum of the micrograph in (D). The position of 4 Å is labeled. See also Figure S1. Structure 2016 24, 1984-1990DOI: (10.1016/j.str.2016.09.008) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Single-Particle 3D Reconstruction of TV Using Affinity Cryo-EM Data (A) Surface representation of TV radially colored according to the provided color key. (B) Gold-standard FSC curves of the overall map (red), the protruding layer (blue), and the shell layer (green). The position of the 0.143 FSC cutoff for resolution estimation is labeled with a dashed line. (C and D) 2ln(Nasud) versus 1/d2 is plotted for Boverall estimation of the affinity cryo-EM data of TV (C) and the cryo-EM dataset of 20S proteasome (EMPIAR-10025) reprocessed using jspr (D), respectively. See also Figures S2 and S3. Structure 2016 24, 1984-1990DOI: (10.1016/j.str.2016.09.008) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Atomic Structural Features Resolved in the ∼2.6-Å Map of TV (A) Densities for a short α helix followed by a short loop. (B and C) Densities for two β strands. (D) Multiple rotameric states identified for Ile residues. (E) One example of distinguishing the correct rotamer (solid arrow) from alternative rotameric states (dashed arrows) for residue Arg140. (F) Water molecules resolved in TV map. Water molecules (pink crosses) were only assigned to densities present in both maps generated using the two half datasets. Hydrogen bonds are shown as dashed lines with the bond length labeled. Structure 2016 24, 1984-1990DOI: (10.1016/j.str.2016.09.008) Copyright © 2016 Elsevier Ltd Terms and Conditions