Fig. 3: IAAG can enrich target protein and overcome preferred orientation problems for atomic resolution cryo-EM. | Communications Biology

Fig. 3: IAAG can enrich target protein and overcome preferred orientation problems for atomic resolution cryo-EM.

From: An immobilized antibody-based affinity grid strategy for on-grid purification of target proteins enables high-resolution cryo-EM

Fig. 3: IAAG can enrich target protein and overcome preferred orientation problems for atomic resolution cryo-EM.

a Cryo-EM image of the PA-tagged apoferritin prepared by the control GO grid, showing the low concentration of the target. b Cryo-EM image of the same sample prepared with IAAG grid, suggesting enrichment of the target protein. (c) 2.5-Å-resolution cryo-EM map of the IAAG enriched apoferritin (left), and high-resolution structure features (right) (model PDB: 6Z6U). d Side and top views of the NZ-1 Fab structure (PDB: 4YNY), with the lysine residues colored in cornflower blue. e Diagram of the His tag and PA tag inserted yeast CCT6 plasmid construct. f Cryo-EM image, reference-free 2D class averages, top/side view particle population analysis for the CCT6-HR-ATP-AlFx on control Quantifoil grid. This demonstrated obvious preferred top-view orientation problems. Top/tilted top views are indicated by cyan square, and side/tilted side views in the yellow square. This color scheme was followed in the rest of this figure. g The corresponding 3D reconstruction (no imposed symmetry) and particle Euler angle distribution for the CCT6-HR-ATP-AlFx on the control Quantifoil grid. h Cryo-EM image, reference-free 2D class averages, top/side view particle population analysis for the CCT6-HR-ATP-AlFx on the IAAG grid. i The corresponding 3D reconstruction (no imposed symmetry) and particle Euler angle distribution for the CCT6-HR-ATP-AlFx on the IAAG grid, which helps overcome the preferred orientation problems.

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