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Identifying and Visualizing Macromolecular Flexibility in Structural Biology

View Article: PubMed Central - PubMed

ABSTRACT

Structural biology comprises a variety of tools to obtain atomic resolution data for the investigation of macromolecules. Conventional structural methodologies including crystallography, NMR and electron microscopy often do not provide sufficient details concerning flexibility and dynamics, even though these aspects are critical for the physiological functions of the systems under investigation. However, the increasing complexity of the molecules studied by structural biology (including large macromolecular assemblies, integral membrane proteins, intrinsically disordered systems, and folding intermediates) continuously demands in-depth analyses of the roles of flexibility and conformational specificity involved in interactions with ligands and inhibitors. The intrinsic difficulties in capturing often subtle but critical molecular motions in biological systems have restrained the investigation of flexible molecules into a small niche of structural biology. Introduction of massive technological developments over the recent years, which include time-resolved studies, solution X-ray scattering, and new detectors for cryo-electron microscopy, have pushed the limits of structural investigation of flexible systems far beyond traditional approaches of NMR analysis. By integrating these modern methods with powerful biophysical and computational approaches such as generation of ensembles of molecular models and selective particle picking in electron microscopy, more feasible investigations of dynamic systems are now possible. Using some prominent examples from recent literature, we review how current structural biology methods can contribute useful data to accurately visualize flexibility in macromolecular structures and understand its important roles in regulation of biological processes.

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Related in: MedlinePlus

Trapping multiple conformations using modern cryo-EM. (A) Three different EM maps obtained from selective classification of the apo gamma secretase cryo micrographs show conformational changes in the transmembrane region of the enzyme complex. Shown are the experimental maps and the three-dimensional structures (obtained from EMDB maps 3238, 3239, 3240, and PDB IDs 5FN3, 5FN4, 5FN5, respectively, Bai et al., 2015b) with soluble nicastrin depicted in green, and the transmembrane region composed of Aph-1, PS1, and Pen-2 components in cyan. Transmembrane helices found in different conformations in the three different classes are shown in blue, red and orange. Arrows indicate the putative movements associated to the rearrangements of the transmembrane helices. (B) Three EM reconstructions relative to identification of multiple conformations in DNA-free and DNA-bound E. coli PolIIIα-clamp-exonuclease-τc micrographs (Fernandez-Leiro et al., 2015). PolIIIα is depicted in cyan, the clamp is shown in green, the exonuclease domain is in blue. DNA is colored in dark gray and is present only in classes 2 and 3. The moving regions, composed of the PolIIIα-tail and τc, are shown in orange and red, respectively (data from EMDB maps 3201, 3198, and 3202). The superposition shows the comparison between the structural models obtained from the DNA-free (class 1) and DNA-bound (class 2) states, shown as cartoon and colored in light and dark blue, respectively (PDB IDs 5FKU and 5FKV). DNA for the bound state is shown in gold. Figure prepared using Chimera (Pettersen et al., 2004).
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Figure 2: Trapping multiple conformations using modern cryo-EM. (A) Three different EM maps obtained from selective classification of the apo gamma secretase cryo micrographs show conformational changes in the transmembrane region of the enzyme complex. Shown are the experimental maps and the three-dimensional structures (obtained from EMDB maps 3238, 3239, 3240, and PDB IDs 5FN3, 5FN4, 5FN5, respectively, Bai et al., 2015b) with soluble nicastrin depicted in green, and the transmembrane region composed of Aph-1, PS1, and Pen-2 components in cyan. Transmembrane helices found in different conformations in the three different classes are shown in blue, red and orange. Arrows indicate the putative movements associated to the rearrangements of the transmembrane helices. (B) Three EM reconstructions relative to identification of multiple conformations in DNA-free and DNA-bound E. coli PolIIIα-clamp-exonuclease-τc micrographs (Fernandez-Leiro et al., 2015). PolIIIα is depicted in cyan, the clamp is shown in green, the exonuclease domain is in blue. DNA is colored in dark gray and is present only in classes 2 and 3. The moving regions, composed of the PolIIIα-tail and τc, are shown in orange and red, respectively (data from EMDB maps 3201, 3198, and 3202). The superposition shows the comparison between the structural models obtained from the DNA-free (class 1) and DNA-bound (class 2) states, shown as cartoon and colored in light and dark blue, respectively (PDB IDs 5FKU and 5FKV). DNA for the bound state is shown in gold. Figure prepared using Chimera (Pettersen et al., 2004).

Mentions: There are numerous examples elucidating the ability of cryo-EM to enable direct analysis of conformational changes in large macromolecular complexes. The structure of the complex of human gamma secretase was determined by implementing new structural refinement methodologies, allowing to “focus” the refinement on a defined region of the protein complex of interest. Such strategy allowed overcoming the issue of structural heterogeneity within the cryo-EM dataset, and allowing characterization of atomic features and side-chain allosteric rearrangements in the active site. The same structural refinement methods enabled understanding how inhibitors of the enzyme complex induce conformational rigidification (Bai et al., 2015b; Figure 2A). In a recent study focusing on processivity in cytoplasmic dynein, cryo-EM showed a wide range of conformations, providing for the first time evidence for extensive flexibility to be essential to the function of this molecular motor (Imai et al., 2015). Recently, five ribosome structures in complex with the viral internal entry sites (IRES) and translocase eEF2 were obtained by accurate classification and particle analysis from a single cryo-EM dataset. These structures, refined to maximum resolutions ranging from 3.5 to 4.2 Å, revealed how the viral molecule progressively translocates in a cap-independent manner from the A to the P sites of the ribosome, and provided an unprecedented view of EF2 dynamics (Abeyrathne et al., 2016). Other fascinating examples of the possibilities of cryo-EM in investigating molecular flexibility are provided by the E. Coli PolIIIα-clamp-exonuclease-τc complex and the hexameric AAA ATPase p97. In the 8 Å resolution structures of DNA-bound and DNA-free states of the PolIII-replisome complex, even if nearly all the proteins composing the complex are flexible enough to hinder crystallography, the cryo-EM structures clearly revealed conformational changes critical for interaction of the replisome with DNA (Fernandez-Leiro et al., 2015; Figure 2B). The cryo-EM micrographs of the hexameric AAA ATPase p97 showed three distinct, co-existing functional states of p97 with differential ATP occupancy per protomer, accompanied by large rearrangements of structural elements in the ATPase fold. Interestingly, the conformations obtained in the cryo-EM reconstructions were never observed in the crystal structures of p97. This example illustrates how multiple 3D reconstructions of distinct conformations of a dynamic macromolecule can be obtained from a single cryo-EM dataset by accurate particle selection and classification after particle picking (Banerjee et al., 2016).


Identifying and Visualizing Macromolecular Flexibility in Structural Biology
Trapping multiple conformations using modern cryo-EM. (A) Three different EM maps obtained from selective classification of the apo gamma secretase cryo micrographs show conformational changes in the transmembrane region of the enzyme complex. Shown are the experimental maps and the three-dimensional structures (obtained from EMDB maps 3238, 3239, 3240, and PDB IDs 5FN3, 5FN4, 5FN5, respectively, Bai et al., 2015b) with soluble nicastrin depicted in green, and the transmembrane region composed of Aph-1, PS1, and Pen-2 components in cyan. Transmembrane helices found in different conformations in the three different classes are shown in blue, red and orange. Arrows indicate the putative movements associated to the rearrangements of the transmembrane helices. (B) Three EM reconstructions relative to identification of multiple conformations in DNA-free and DNA-bound E. coli PolIIIα-clamp-exonuclease-τc micrographs (Fernandez-Leiro et al., 2015). PolIIIα is depicted in cyan, the clamp is shown in green, the exonuclease domain is in blue. DNA is colored in dark gray and is present only in classes 2 and 3. The moving regions, composed of the PolIIIα-tail and τc, are shown in orange and red, respectively (data from EMDB maps 3201, 3198, and 3202). The superposition shows the comparison between the structural models obtained from the DNA-free (class 1) and DNA-bound (class 2) states, shown as cartoon and colored in light and dark blue, respectively (PDB IDs 5FKU and 5FKV). DNA for the bound state is shown in gold. Figure prepared using Chimera (Pettersen et al., 2004).
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Figure 2: Trapping multiple conformations using modern cryo-EM. (A) Three different EM maps obtained from selective classification of the apo gamma secretase cryo micrographs show conformational changes in the transmembrane region of the enzyme complex. Shown are the experimental maps and the three-dimensional structures (obtained from EMDB maps 3238, 3239, 3240, and PDB IDs 5FN3, 5FN4, 5FN5, respectively, Bai et al., 2015b) with soluble nicastrin depicted in green, and the transmembrane region composed of Aph-1, PS1, and Pen-2 components in cyan. Transmembrane helices found in different conformations in the three different classes are shown in blue, red and orange. Arrows indicate the putative movements associated to the rearrangements of the transmembrane helices. (B) Three EM reconstructions relative to identification of multiple conformations in DNA-free and DNA-bound E. coli PolIIIα-clamp-exonuclease-τc micrographs (Fernandez-Leiro et al., 2015). PolIIIα is depicted in cyan, the clamp is shown in green, the exonuclease domain is in blue. DNA is colored in dark gray and is present only in classes 2 and 3. The moving regions, composed of the PolIIIα-tail and τc, are shown in orange and red, respectively (data from EMDB maps 3201, 3198, and 3202). The superposition shows the comparison between the structural models obtained from the DNA-free (class 1) and DNA-bound (class 2) states, shown as cartoon and colored in light and dark blue, respectively (PDB IDs 5FKU and 5FKV). DNA for the bound state is shown in gold. Figure prepared using Chimera (Pettersen et al., 2004).
Mentions: There are numerous examples elucidating the ability of cryo-EM to enable direct analysis of conformational changes in large macromolecular complexes. The structure of the complex of human gamma secretase was determined by implementing new structural refinement methodologies, allowing to “focus” the refinement on a defined region of the protein complex of interest. Such strategy allowed overcoming the issue of structural heterogeneity within the cryo-EM dataset, and allowing characterization of atomic features and side-chain allosteric rearrangements in the active site. The same structural refinement methods enabled understanding how inhibitors of the enzyme complex induce conformational rigidification (Bai et al., 2015b; Figure 2A). In a recent study focusing on processivity in cytoplasmic dynein, cryo-EM showed a wide range of conformations, providing for the first time evidence for extensive flexibility to be essential to the function of this molecular motor (Imai et al., 2015). Recently, five ribosome structures in complex with the viral internal entry sites (IRES) and translocase eEF2 were obtained by accurate classification and particle analysis from a single cryo-EM dataset. These structures, refined to maximum resolutions ranging from 3.5 to 4.2 Å, revealed how the viral molecule progressively translocates in a cap-independent manner from the A to the P sites of the ribosome, and provided an unprecedented view of EF2 dynamics (Abeyrathne et al., 2016). Other fascinating examples of the possibilities of cryo-EM in investigating molecular flexibility are provided by the E. Coli PolIIIα-clamp-exonuclease-τc complex and the hexameric AAA ATPase p97. In the 8 Å resolution structures of DNA-bound and DNA-free states of the PolIII-replisome complex, even if nearly all the proteins composing the complex are flexible enough to hinder crystallography, the cryo-EM structures clearly revealed conformational changes critical for interaction of the replisome with DNA (Fernandez-Leiro et al., 2015; Figure 2B). The cryo-EM micrographs of the hexameric AAA ATPase p97 showed three distinct, co-existing functional states of p97 with differential ATP occupancy per protomer, accompanied by large rearrangements of structural elements in the ATPase fold. Interestingly, the conformations obtained in the cryo-EM reconstructions were never observed in the crystal structures of p97. This example illustrates how multiple 3D reconstructions of distinct conformations of a dynamic macromolecule can be obtained from a single cryo-EM dataset by accurate particle selection and classification after particle picking (Banerjee et al., 2016).

View Article: PubMed Central - PubMed

ABSTRACT

Structural biology comprises a variety of tools to obtain atomic resolution data for the investigation of macromolecules. Conventional structural methodologies including crystallography, NMR and electron microscopy often do not provide sufficient details concerning flexibility and dynamics, even though these aspects are critical for the physiological functions of the systems under investigation. However, the increasing complexity of the molecules studied by structural biology (including large macromolecular assemblies, integral membrane proteins, intrinsically disordered systems, and folding intermediates) continuously demands in-depth analyses of the roles of flexibility and conformational specificity involved in interactions with ligands and inhibitors. The intrinsic difficulties in capturing often subtle but critical molecular motions in biological systems have restrained the investigation of flexible molecules into a small niche of structural biology. Introduction of massive technological developments over the recent years, which include time-resolved studies, solution X-ray scattering, and new detectors for cryo-electron microscopy, have pushed the limits of structural investigation of flexible systems far beyond traditional approaches of NMR analysis. By integrating these modern methods with powerful biophysical and computational approaches such as generation of ensembles of molecular models and selective particle picking in electron microscopy, more feasible investigations of dynamic systems are now possible. Using some prominent examples from recent literature, we review how current structural biology methods can contribute useful data to accurately visualize flexibility in macromolecular structures and understand its important roles in regulation of biological processes.

No MeSH data available.


Related in: MedlinePlus