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

Zhang X, Zhang L, Tong H, Peng B, Rames MJ, Zhang S, Ren G - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA [2] Department of Applied Physics, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

3D reconstructions of an individual IgG antibody particle by individual-particle electron tomography (IPET). a, An individual IgG antibody particle was targeted and selected from a tilt series of OpNS-ET micrographs (81 tilt angles, tilt angles from −60° to +60°, in steps of 1.5°). Seven representative tilted views of the targeted particle (selected from 81 tilt micrographs after CTF correction) are displayed in the first column from the left. During the process of IPET reconstruction, the tilting images were gradually aligned to a common center (two translational parameters, x and y) for 3D reconstruction via an iterative refinement process. In round 1 (rd. 1) iterations, a set of automatically generated circular soft-boundary masks was sequentially used. In round 2 (rd. 2), an automatically generated particle-shaped soft-masks were sequentially applied. The round 3 (rd. 3) repeated the rd. 2 iteration with its final mask under an interpolation condition. The projections of the intermediate and final 3D reconstructions at the corresponding tilting angles are displayed beside the raw images in the next six columns. b, The final 3D density map of the targeted individual antibody particle reconstructed by IPET. c, The final map (displayed in two iso-surfaces) provided a constraint to dock the crystal structure flexibly to achieve a new conformation of antibody via targeted molecular dynamics (TMD) simulations. d, Fourier shell correlation (FSC) curve were computed by calculating the cross-correlation of the two density maps reconstructed independently from the two halves (odd and even numbers) of original tilting images (before alignment). The spatial frequency at which FSC fell below 0.5 (instead of 0.143) was modestly used to represent the resolution of the final 3D reconstruction. e, Five snapshot images during TMD simulation illustrated the process of flexibly docking the crystal structure (PDB: 1IGT) into the IPET density map to achieve a new conformation of antibody. During this process, the domain crystal structure remained unchanged (as a rigid body). However, the two flexible linkers were allowed to change in structure, but still maintained proper chemical geometry and bonds. The scale bar was 50 Å.
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f2: 3D reconstructions of an individual IgG antibody particle by individual-particle electron tomography (IPET). a, An individual IgG antibody particle was targeted and selected from a tilt series of OpNS-ET micrographs (81 tilt angles, tilt angles from −60° to +60°, in steps of 1.5°). Seven representative tilted views of the targeted particle (selected from 81 tilt micrographs after CTF correction) are displayed in the first column from the left. During the process of IPET reconstruction, the tilting images were gradually aligned to a common center (two translational parameters, x and y) for 3D reconstruction via an iterative refinement process. In round 1 (rd. 1) iterations, a set of automatically generated circular soft-boundary masks was sequentially used. In round 2 (rd. 2), an automatically generated particle-shaped soft-masks were sequentially applied. The round 3 (rd. 3) repeated the rd. 2 iteration with its final mask under an interpolation condition. The projections of the intermediate and final 3D reconstructions at the corresponding tilting angles are displayed beside the raw images in the next six columns. b, The final 3D density map of the targeted individual antibody particle reconstructed by IPET. c, The final map (displayed in two iso-surfaces) provided a constraint to dock the crystal structure flexibly to achieve a new conformation of antibody via targeted molecular dynamics (TMD) simulations. d, Fourier shell correlation (FSC) curve were computed by calculating the cross-correlation of the two density maps reconstructed independently from the two halves (odd and even numbers) of original tilting images (before alignment). The spatial frequency at which FSC fell below 0.5 (instead of 0.143) was modestly used to represent the resolution of the final 3D reconstruction. e, Five snapshot images during TMD simulation illustrated the process of flexibly docking the crystal structure (PDB: 1IGT) into the IPET density map to achieve a new conformation of antibody. During this process, the domain crystal structure remained unchanged (as a rigid body). However, the two flexible linkers were allowed to change in structure, but still maintained proper chemical geometry and bonds. The scale bar was 50 Å.

Mentions: Although the signal to noise ratios (SNR) of an individual antibody particle in each tilt image (from −60° to + 60° in a step of 1.5°) were only ~0.1 to ~0.26 in the raw micrographs, the overall shape of each individual antibody particle was still visible (Supplementary Video). The images of a targeted antibody particle were iteratively aligned to a global center for an ab-inito 3D reconstruction with IPET (Fig. 2 and Supplementary video). During the iterations, the SNRs in the 3D projections were gradually increased to ~1.3 to ~1.8 in the final 3D reconstruction. The dual iso-surfaces of the final 3D reconstruction (displayed in their contained volumes corresponding to 0.6 and 1.6 times of the antibody mass of ~150 kDa) showed three globular densities (two of which were ~60 Å and one ~70 Å in diameter), forming a “Y” shaped structure with an overall diameter of ~150–180 Å. (Fig. 2b).


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

Zhang X, Zhang L, Tong H, Peng B, Rames MJ, Zhang S, Ren G - Sci Rep (2015)

3D reconstructions of an individual IgG antibody particle by individual-particle electron tomography (IPET). a, An individual IgG antibody particle was targeted and selected from a tilt series of OpNS-ET micrographs (81 tilt angles, tilt angles from −60° to +60°, in steps of 1.5°). Seven representative tilted views of the targeted particle (selected from 81 tilt micrographs after CTF correction) are displayed in the first column from the left. During the process of IPET reconstruction, the tilting images were gradually aligned to a common center (two translational parameters, x and y) for 3D reconstruction via an iterative refinement process. In round 1 (rd. 1) iterations, a set of automatically generated circular soft-boundary masks was sequentially used. In round 2 (rd. 2), an automatically generated particle-shaped soft-masks were sequentially applied. The round 3 (rd. 3) repeated the rd. 2 iteration with its final mask under an interpolation condition. The projections of the intermediate and final 3D reconstructions at the corresponding tilting angles are displayed beside the raw images in the next six columns. b, The final 3D density map of the targeted individual antibody particle reconstructed by IPET. c, The final map (displayed in two iso-surfaces) provided a constraint to dock the crystal structure flexibly to achieve a new conformation of antibody via targeted molecular dynamics (TMD) simulations. d, Fourier shell correlation (FSC) curve were computed by calculating the cross-correlation of the two density maps reconstructed independently from the two halves (odd and even numbers) of original tilting images (before alignment). The spatial frequency at which FSC fell below 0.5 (instead of 0.143) was modestly used to represent the resolution of the final 3D reconstruction. e, Five snapshot images during TMD simulation illustrated the process of flexibly docking the crystal structure (PDB: 1IGT) into the IPET density map to achieve a new conformation of antibody. During this process, the domain crystal structure remained unchanged (as a rigid body). However, the two flexible linkers were allowed to change in structure, but still maintained proper chemical geometry and bonds. The scale bar was 50 Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4419541&req=5

f2: 3D reconstructions of an individual IgG antibody particle by individual-particle electron tomography (IPET). a, An individual IgG antibody particle was targeted and selected from a tilt series of OpNS-ET micrographs (81 tilt angles, tilt angles from −60° to +60°, in steps of 1.5°). Seven representative tilted views of the targeted particle (selected from 81 tilt micrographs after CTF correction) are displayed in the first column from the left. During the process of IPET reconstruction, the tilting images were gradually aligned to a common center (two translational parameters, x and y) for 3D reconstruction via an iterative refinement process. In round 1 (rd. 1) iterations, a set of automatically generated circular soft-boundary masks was sequentially used. In round 2 (rd. 2), an automatically generated particle-shaped soft-masks were sequentially applied. The round 3 (rd. 3) repeated the rd. 2 iteration with its final mask under an interpolation condition. The projections of the intermediate and final 3D reconstructions at the corresponding tilting angles are displayed beside the raw images in the next six columns. b, The final 3D density map of the targeted individual antibody particle reconstructed by IPET. c, The final map (displayed in two iso-surfaces) provided a constraint to dock the crystal structure flexibly to achieve a new conformation of antibody via targeted molecular dynamics (TMD) simulations. d, Fourier shell correlation (FSC) curve were computed by calculating the cross-correlation of the two density maps reconstructed independently from the two halves (odd and even numbers) of original tilting images (before alignment). The spatial frequency at which FSC fell below 0.5 (instead of 0.143) was modestly used to represent the resolution of the final 3D reconstruction. e, Five snapshot images during TMD simulation illustrated the process of flexibly docking the crystal structure (PDB: 1IGT) into the IPET density map to achieve a new conformation of antibody. During this process, the domain crystal structure remained unchanged (as a rigid body). However, the two flexible linkers were allowed to change in structure, but still maintained proper chemical geometry and bonds. The scale bar was 50 Å.
Mentions: Although the signal to noise ratios (SNR) of an individual antibody particle in each tilt image (from −60° to + 60° in a step of 1.5°) were only ~0.1 to ~0.26 in the raw micrographs, the overall shape of each individual antibody particle was still visible (Supplementary Video). The images of a targeted antibody particle were iteratively aligned to a global center for an ab-inito 3D reconstruction with IPET (Fig. 2 and Supplementary video). During the iterations, the SNRs in the 3D projections were gradually increased to ~1.3 to ~1.8 in the final 3D reconstruction. The dual iso-surfaces of the final 3D reconstruction (displayed in their contained volumes corresponding to 0.6 and 1.6 times of the antibody mass of ~150 kDa) showed three globular densities (two of which were ~60 Å and one ~70 Å in diameter), forming a “Y” shaped structure with an overall diameter of ~150–180 Å. (Fig. 2b).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA [2] Department of Applied Physics, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus