3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography

Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.


Supplementary Figure 5 | Detailed procedures of the fourth representative IPET reconstruction.
Detailed IPET reconstruction of particle No. 4 was presented here. a, Seven representative tilted views of an individual IgG antibody particle shown in the first column (SNR: 0.16), followed by the corresponding projections of 3D reconstruction from major iterations (second to fifth columns), with the final result from each tilting angle shown in the last column. b, The finalized 3D reconstruction image (SNR: 1.31) of a targeted individual antibody eventually obtained after the above process. c, 3D density map and pseudo structure. d, FSC curve of IPET reconstruction. e, Five snapshot images illustrated the ease of TMD simulation. Molecular Dynamic simulation was used to steer the three domains with a force calculated to match ultimately the three domain density maps in an aqueous environment. The scale bar was 50 Å.
Supplementary Figure 6 | Detailed procedures of the fifth representative IPET reconstruction. Detailed IPET reconstruction of particle No. 5 was presented here. a, Seven representative tilted views of an individual IgG antibody particle shown in the first column (SNR: 0.15), followed by the corresponding projections of 3D reconstruction from major iterations (second to fifth columns), with the final result from each tilting angle shown in the last column. b, The finalized 3D reconstruction image (SNR: 1.55) of a targeted individual antibody eventually obtained after the above process. c, 3D density map and pseudo structure. d, FSC curve of IPET reconstruction. e, Five snapshot images illustrated the ease of TMD simulation. Molecular Dynamic simulation was used to steer the three domains with a force calculated to match ultimately the three domain density maps in an aqueous environment. The scale bar was 50 Å.

Supplementary Figure 7 | Detailed procedures of the sixth representative IPET reconstruction.
Detailed IPET reconstruction of particle No. 6 was presented here. a, Seven representative tilted views of an individual IgG antibody particle shown in the first column (SNR: 0.23), followed by the corresponding projections of 3D reconstruction from major iterations (second to fifth columns), with the final result from each tilting angle shown in the last column. b, The finalized 3D reconstruction image (SNR: 1.73) of a targeted individual antibody eventually obtained after the above process. c, 3D density map and pseudo structure. d, FSC curve of IPET reconstruction. e, Five snapshot images illustrated the ease of TMD simulation. Molecular Dynamic simulation was used to steer the three domains with a force calculated to match ultimately the three domain density maps in an aqueous environment. The scale bar was 50 Å.

Supplementary Figure 8 | Detailed procedures of the seventh representative IPET reconstruction.
Detailed IPET reconstruction of particle No. 7 was presented here. a, Seven representative tilted views of an individual IgG antibody particle shown in the first column (SNR: 0.11), followed by the corresponding projections of 3D reconstruction from major iterations (second to fifth columns), with the final result from each tilting angle shown in the last column. b, The finalized 3D reconstruction image (SNR: 1.23) of a targeted individual antibody eventually obtained after the above process. c, 3D density map and pseudo structure. d, FSC curve of IPET reconstruction. e, Five snapshot images illustrated the ease of TMD simulation. Molecular Dynamic simulation was used to steer the three domains with a force calculated to match ultimately the three domain density maps in an aqueous environment. The scale bar was 50 Å.

Supplementary Figure 9 | Detailed procedures of the eighth representative IPET reconstruction.
Detailed IPET reconstruction of particle No. 8 was presented here. a, Seven representative tilted views of an individual IgG antibody particle shown in the first column (SNR: 0.13), followed by the corresponding projections of 3D reconstruction from major iterations (second to fifth columns), with the final result from each tilting angle shown in the last column. b, The finalized 3D reconstruction image (SNR: 1.35) of a targeted individual antibody eventually obtained after the above process. c, 3D density map and pseudo structure. d, FSC curve of IPET reconstruction. e, Five snapshot images illustrated the ease of TMD simulation. Molecular Dynamic simulation was used to steer the three domains with a force calculated to match ultimately the three domain density maps in an aqueous environment. The scale bar was 50 Å.

Supplementary Figure 10 | Detailed procedures of the ninth representative IPET reconstruction.
Detailed IPET reconstruction of particle No. 9 was presented here. a, Seven representative tilted views of an individual IgG antibody particle shown in the first column (SNR: 0.17), followed by the corresponding projections of 3D reconstruction from major iterations (second to fifth columns), with the final result from each tilting angle shown in the last column. b, The finalized 3D reconstruction image (SNR: 1.42) of a targeted individual antibody eventually obtained after the above process. c, 3D density map and pseudo structure. d, FSC curve of IPET reconstruction. e, Five snapshot images illustrated the ease of TMD simulation. Molecular Dynamic simulation was used to steer the three domains with a force calculated to match ultimately the three domain density maps in an aqueous environment. The scale bar was 50 Å. Figure 11 | Detailed procedures of the tenth representative IPET reconstruction. Detailed IPET reconstruction of particle No. 10 was presented here. a, Seven representative tilted views of an individual IgG antibody particle shown in the first column (SNR: 0.26), followed by the corresponding projections of 3D reconstruction from major iterations (second to fifth columns), with the final result from each tilting angle shown in the last column. b, The finalized 3D reconstruction image (SNR: 1.56) of a targeted individual antibody eventually obtained after the above process. c, 3D density map and pseudo structure. d, FSC curve of IPET reconstruction. e, Five snapshot images illustrated the ease of TMD simulation. Molecular Dynamic simulation was used to steer the three domains with a force calculated to match ultimately the three domain density maps in an aqueous environment. The scale bar was 50 Å.

Supplementary Figure 12 | Detailed procedures of the eleventh representative IPET reconstruction.
Detailed IPET reconstruction of particle No. 11 was presented here. a, Seven representative tilted views of an individual IgG antibody particle shown in the first column (SNR: 0.18), followed by the corresponding projections of 3D reconstruction from major iterations (second to fifth columns), with the final result from each tilting angle shown in the last column. b, The finalized 3D reconstruction image (SNR: 1.45) of a targeted individual antibody eventually obtained after the above process. c, 3D density map and pseudo structure. d, FSC curve of IPET reconstruction. e, Five snapshot images illustrated the ease of TMD simulation. Molecular Dynamic simulation was used to steer the three domains with a force calculated to match ultimately the three domain density maps in an aqueous environment. The scale bar was 50 Å.

Supplementary Figure 13 | Detailed procedures of the twelfth representative IPET reconstruction.
Detailed IPET reconstruction of particle No. 12 was presented here. a, Seven representative tilted views of an individual IgG antibody particle shown in the first column (SNR: 0.24), followed by the corresponding projections of 3D reconstruction from major iterations (second to fifth columns), with the final result from each tilting angle shown in the last column. b, The finalized 3D reconstruction image (SNR: 1.84) of a targeted individual antibody eventually obtained after the above process. c, 3D density map and pseudo structure. d, FSC curve of IPET reconstruction. e, Five snapshot images illustrated the ease of TMD simulation. Molecular Dynamic simulation was used to steer the three domains with a force calculated to match ultimately the three domain density maps in an aqueous environment. The scale bar was 50 Å. Figure 14 | Detailed procedures of the thirteenth representative IPET reconstruction. Detailed IPET reconstruction of particle No. 120 was presented here. a, Seven representative tilted views of an individual IgG antibody particle shown in the first column (SNR: 0.26), followed by the corresponding projections of 3D reconstruction from major iterations (second to fifth columns), with the final result from each tilting angle shown in the last column. b, The finalized 3D reconstruction image (SNR: 1.50) of a targeted individual antibody eventually obtained after the above process. c, 3D density map and pseudo structure. d, FSC curve of IPET reconstruction. e, Five snapshot images illustrated the ease of TMD simulation. Molecular Dynamic simulation was used to steer the three domains with a force calculated to match ultimately the three domain density maps in an aqueous environment. The scale bar was 50 Å. Figure 15 | Averaging the density maps of 120 3D density maps of IgG1 antibody. As a single-particle electron tomography method, all 120 IPET 3D density maps were aligned and averaged. The averaged map showed fuzzy domains as similar to those from the 2D class averages and single-particle reconstructions. All density maps were shifted and rotated with respect to the respective domain/linker positions described by vector map in a, and averaged.