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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

A zoom-in gallery of eleven representative 3D density maps of antibody reconstructed by IPET. 11 representative 3D density maps (selected from 120 final 3D density maps of antibody) were displayed in two contour levels. The volume of the inner iso-surface map corresponded to 0.6 times of an IgG1 molecular mass (150kDa), while the volume of outer iso-surface map corresponded to 1.6 times of its mass. By using the EM density maps as constraints, the crystal structure was flexibly docked onto these EM density maps by TMD simulations. During this process, the domain crystal structure remained unchanged; however, two linking loops were changed under condition of energy minima. A total of twelve conformations were achieved. PDB was represented by ribbon and chains were shown by colors.
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f4: A zoom-in gallery of eleven representative 3D density maps of antibody reconstructed by IPET. 11 representative 3D density maps (selected from 120 final 3D density maps of antibody) were displayed in two contour levels. The volume of the inner iso-surface map corresponded to 0.6 times of an IgG1 molecular mass (150kDa), while the volume of outer iso-surface map corresponded to 1.6 times of its mass. By using the EM density maps as constraints, the crystal structure was flexibly docked onto these EM density maps by TMD simulations. During this process, the domain crystal structure remained unchanged; however, two linking loops were changed under condition of energy minima. A total of twelve conformations were achieved. PDB was represented by ribbon and chains were shown by colors.

Mentions: Through particle-by-particle 3D reconstructions by IPET, a total of 120 antibody particles were reconstructed (Fig. 3, 4, 5a and Supplementary Fig. 4-14) from a pool of ~300 targeted particles (selected from ~18 sets of tomography data). Certain particles were excluded due to either particle-particle overlapping at certain tilt angles, missing tilted views, unevenly stained surrounding backgrounds, or indistinguishable Fab/Fc domains. The 120 reconstructions (the resolutions of which were all better than ~20 Å) at two contour levels were displayed after alignment based on their Fc domains, and then put into arrays (Fig. 5a). The Fc domain of each antibody particle was similar in size and shape to that of the crystal structure, while the Fab domains varied widely in their locations and orientations. By flexibly docking the crystal structure into the EM density maps via TMD simulations, we obtained a total of 120 new conformations (Fig. 2c, 3c, 4, 5b, Supplementary Fig. 4-14e and Supplementary Video). ~50% of the both Fc and Fab domains from the EM maps did not completely cover the crystal structure, or were partially different from the crystal structure (Fig. 5b). This might be due to conformational flexibly of domain itself, uneven negative-staining, noise, or reconstruction errors. However, considering that these mismatched portions generally contained less than 25% of the domain volume, these defects did not affect the determination of domain overall location and orientation substantially, and the conformations of each antibody could still be defined from these maps.


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)

A zoom-in gallery of eleven representative 3D density maps of antibody reconstructed by IPET. 11 representative 3D density maps (selected from 120 final 3D density maps of antibody) were displayed in two contour levels. The volume of the inner iso-surface map corresponded to 0.6 times of an IgG1 molecular mass (150kDa), while the volume of outer iso-surface map corresponded to 1.6 times of its mass. By using the EM density maps as constraints, the crystal structure was flexibly docked onto these EM density maps by TMD simulations. During this process, the domain crystal structure remained unchanged; however, two linking loops were changed under condition of energy minima. A total of twelve conformations were achieved. PDB was represented by ribbon and chains were shown by colors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: A zoom-in gallery of eleven representative 3D density maps of antibody reconstructed by IPET. 11 representative 3D density maps (selected from 120 final 3D density maps of antibody) were displayed in two contour levels. The volume of the inner iso-surface map corresponded to 0.6 times of an IgG1 molecular mass (150kDa), while the volume of outer iso-surface map corresponded to 1.6 times of its mass. By using the EM density maps as constraints, the crystal structure was flexibly docked onto these EM density maps by TMD simulations. During this process, the domain crystal structure remained unchanged; however, two linking loops were changed under condition of energy minima. A total of twelve conformations were achieved. PDB was represented by ribbon and chains were shown by colors.
Mentions: Through particle-by-particle 3D reconstructions by IPET, a total of 120 antibody particles were reconstructed (Fig. 3, 4, 5a and Supplementary Fig. 4-14) from a pool of ~300 targeted particles (selected from ~18 sets of tomography data). Certain particles were excluded due to either particle-particle overlapping at certain tilt angles, missing tilted views, unevenly stained surrounding backgrounds, or indistinguishable Fab/Fc domains. The 120 reconstructions (the resolutions of which were all better than ~20 Å) at two contour levels were displayed after alignment based on their Fc domains, and then put into arrays (Fig. 5a). The Fc domain of each antibody particle was similar in size and shape to that of the crystal structure, while the Fab domains varied widely in their locations and orientations. By flexibly docking the crystal structure into the EM density maps via TMD simulations, we obtained a total of 120 new conformations (Fig. 2c, 3c, 4, 5b, Supplementary Fig. 4-14e and Supplementary Video). ~50% of the both Fc and Fab domains from the EM maps did not completely cover the crystal structure, or were partially different from the crystal structure (Fig. 5b). This might be due to conformational flexibly of domain itself, uneven negative-staining, noise, or reconstruction errors. However, considering that these mismatched portions generally contained less than 25% of the domain volume, these defects did not affect the determination of domain overall location and orientation substantially, and the conformations of each antibody could still be defined from these maps.

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