Fig 1.
The antibody structure and variability.
(A) X-ray crystal structures of three full length antibodies: mouse IgG2 in blue (PDB code 1IGT) [3], human IgG1 in red (1HZH) [4], and mouse IgG1 in green (1IGY) [5], superposed on all aligned Cα atoms from all three domains (left) for the overall shape comparison or only Fc domains (right) to highlight the differences in Fab domains. The pairwise Cα RMSD values range from 20 to 34 Å with an average of 27.6 Å. The pairwise Cα RMSD values of the Fab domains range from 1.1 to 3.9 Å when superposed only on the Fab domain residues, with an average of 2.0 Å. The pairwise Cα RMSD values of the Fc domains range from 2.2 to 2.4 Å when superposed only on the Fc domain residues. Superposition was done using MOE 2014.09 [6]. (B) An example to show the definition of the domain angles between every two domains measured from 3D structures as described by Zhang et. al. [2]. The lines follow the longest axis of each domain.
Fig 2.
The effect of MET antibody isotypes on pAKT in Caki-1 cells.
(A) The humanized IgG4 MET antibody (LY2875358), hIgG4 and mIgG1 induce weak phosphorylation of pan-AKT as compared to the strong phosphorylation of pan-AKT by agonist antibody 5D5 and HGF [12]. (B) Comparison of IgG1 (purple), IgG2 (blue) and IgG4 MET antibodies (red). (C) The sequences in the hinges. The numbering of the first residue is shown in each sequence. The inter-heavy-chain disulfide bonded cysteine residues are indicated in yellow boxes.
Fig 3.
Examples of EM image thumbnails.
An EM micrograph with a number of IgG1 particles (left) and EM 2D class average images (right). Not all 2D class averages contained recognizable Y/T-shaped antibody particles; those marked by a red “X” were not used in modeling.
Fig 4.
Observed class averages, resulting 3D models, and class averages computed from the models.
Images of observed 2D class average (first row), class averages computed from the resulting 3D models (second row) and the ribbon views from the 3D model (light chains in green and magenta, heavy chains in yellow and cyan, glycoside heavy atoms in red), and the models (second row). (A) IgG1: image dimension 160x160 pixels, 2.0 Å/pixel. (B) IgG2: image dimension 160x160 pixels, 2.0 Å/pixel. (C) IgG4: image dimension 106x106 pixels, 3.0 Å/pixel. (D) IgG4-MET antigen complex: image dimension 160x160 pixels, 3.24 Å/pixel.
Fig 5.
Distributions of pairwise RMSD values for IgG1, IgG2, IgG4 and IgG4- MET complex models.
The average values of pairwise RMSD are indicated by the gray vertical solid bars. The 95% confidence intervals of the average are indicated by the black vertical dashed bars. The plots were created using TIBCO Spotfire 6.5.3.[16]
Fig 6.
EM2D scores of all conformations and their RMSD values to the highest scoring conformation.
The conformations are binned into groups of 1 Å size according to the RMSD values. The standard error is shown as the error bar for each bin. The samples are (A) IgG1, (B) IgG2, (C) IgG4, and (D) IgG4 antigen complex.
Table 1.
Flexibility of domain arrangements.
The differences in average values of the domain angles are not statistically significant at 95% confidence level (Figure B in S1 File).
Fig 7.
Flowchart of integrative multi-state modeling.