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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

Negative-staining images and single-particle three-dimensional (3D) reconstructions of IgG1 antibody. a, A survey of various antibody particles (dashed circles) prepared by optimized negative-staining. The “Y”-shaped particles contained three globular circular or oval domains. b, Eight representative individual particles of antibody. c, Eight representative reference-free class averages (selected from 80 classes) showed some domains (arrows) were fuzzy or blurry in density. The scale bar was 100 Å.
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f1: Negative-staining images and single-particle three-dimensional (3D) reconstructions of IgG1 antibody. a, A survey of various antibody particles (dashed circles) prepared by optimized negative-staining. The “Y”-shaped particles contained three globular circular or oval domains. b, Eight representative individual particles of antibody. c, Eight representative reference-free class averages (selected from 80 classes) showed some domains (arrows) were fuzzy or blurry in density. The scale bar was 100 Å.

Mentions: Imaging of IgG1 antibody (molecular mass ~150 kDa) was performed by optimized negative-staining (OpNS) EM technique1415, instead of electron cryo-microscopy (cryo-EM). Cryo-EM often poses a challenge in imaging proteins with molecular masses less than 200 kDa. The survey image (after being Gaussian low-pass filtered to 20Å) showed evenly distributed antibodies having a “Y” shape with dimensions of ~150–180 Å (circles in Fig. 1a, and squares in Supplementary Video). Most antibody particles contained three “ring-shaped” domains of ~55–75 Å in diameter (Fig. 1b and Supplementary Fig. 1a), which corresponded to two Fab domains and one Fc domain. The domain sizes and shapes were similar to those of the corresponding crystal structures (PDB entry, 1IGT16, 1IGY17, 1HZH18), suggesting that antibody domains could directly be visualized by OpNS EM technique. The reference-free class averages from 11,373 particles confirmed a “Y”-shape structure (Fig. 1c). However, about half of the class averages were fuzzy or blurry in one or two domains. The blurry domains were due to the protein containing flexible domains (arrows indicated in Fig. 1c and Supplementary Fig. 1b), suggesting the protein was unsuitable for single-particle 3D reconstruction. However, if we ignored these flexibilities and enforced to conduct the conventional approach for 3-dimensional (3D) reconstruction, i.e. single-particle reconstruction methods, the 3D reconstructions refined from two sets of initial models showed the final 3D reconstructions contained artifacts in domain structures such as domain size (detailed description in the discussion section and Supplementary Fig. 2).


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)

Negative-staining images and single-particle three-dimensional (3D) reconstructions of IgG1 antibody. a, A survey of various antibody particles (dashed circles) prepared by optimized negative-staining. The “Y”-shaped particles contained three globular circular or oval domains. b, Eight representative individual particles of antibody. c, Eight representative reference-free class averages (selected from 80 classes) showed some domains (arrows) were fuzzy or blurry in density. The scale bar was 100 Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Negative-staining images and single-particle three-dimensional (3D) reconstructions of IgG1 antibody. a, A survey of various antibody particles (dashed circles) prepared by optimized negative-staining. The “Y”-shaped particles contained three globular circular or oval domains. b, Eight representative individual particles of antibody. c, Eight representative reference-free class averages (selected from 80 classes) showed some domains (arrows) were fuzzy or blurry in density. The scale bar was 100 Å.
Mentions: Imaging of IgG1 antibody (molecular mass ~150 kDa) was performed by optimized negative-staining (OpNS) EM technique1415, instead of electron cryo-microscopy (cryo-EM). Cryo-EM often poses a challenge in imaging proteins with molecular masses less than 200 kDa. The survey image (after being Gaussian low-pass filtered to 20Å) showed evenly distributed antibodies having a “Y” shape with dimensions of ~150–180 Å (circles in Fig. 1a, and squares in Supplementary Video). Most antibody particles contained three “ring-shaped” domains of ~55–75 Å in diameter (Fig. 1b and Supplementary Fig. 1a), which corresponded to two Fab domains and one Fc domain. The domain sizes and shapes were similar to those of the corresponding crystal structures (PDB entry, 1IGT16, 1IGY17, 1HZH18), suggesting that antibody domains could directly be visualized by OpNS EM technique. The reference-free class averages from 11,373 particles confirmed a “Y”-shape structure (Fig. 1c). However, about half of the class averages were fuzzy or blurry in one or two domains. The blurry domains were due to the protein containing flexible domains (arrows indicated in Fig. 1c and Supplementary Fig. 1b), suggesting the protein was unsuitable for single-particle 3D reconstruction. However, if we ignored these flexibilities and enforced to conduct the conventional approach for 3-dimensional (3D) reconstruction, i.e. single-particle reconstruction methods, the 3D reconstructions refined from two sets of initial models showed the final 3D reconstructions contained artifacts in domain structures such as domain size (detailed description in the discussion section and Supplementary Fig. 2).

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