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Towards an integrative structural biology approach: combining Cryo-TEM, X-ray crystallography, and NMR.

Lengyel J, Hnath E, Storms M, Wohlfarth T - J. Struct. Funct. Genomics (2014)

Bottom Line: In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques.Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable.Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.

View Article: PubMed Central - PubMed

Affiliation: FEI Company, 5350 N.E. Dawson Creek Drive, Hillsboro, OR, 97124, USA, Jeffrey.lengyel@fei.com.

ABSTRACT
Cryo-transmission electron microscopy (Cryo-TEM) and particularly single particle analysis is rapidly becoming the premier method for determining the three-dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques. While Cryo-TEM was once thought of as a low resolution structural technique, the method is currently capable of generating nearly atomic resolution structures on a routine basis. Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable. Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.

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Using cryo-tomographic analysis of individual, native “fullerene” cone HIV-1 capsid, combined with the high resolution Cryo-TEM hexamer structures an all-atom molecular dynamics HIV-1 capsid model was created. This model highlights the threefold capsid protein C-terminal domain as an attractive therapeutic target. a Slice through cryo-tomographic volume of an individual mature capsid. Red arrows highlight CA pentamers. b The tomographic density matches the shape and size of the capsid, shown by the overlay of densities from the segmented capsid and the fullerene model (yellow). c Final molecular dynamics equilibrated all-atom capsid model. Images kindly provided by Peijun Zhang Univ. of Pittsburgh. Adapted from [22]. EMDB Accession codes: EMD-5582, EMD-5639; Fitted PDB IDs: 3J34, 3J4F, 3J3Y
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Fig5: Using cryo-tomographic analysis of individual, native “fullerene” cone HIV-1 capsid, combined with the high resolution Cryo-TEM hexamer structures an all-atom molecular dynamics HIV-1 capsid model was created. This model highlights the threefold capsid protein C-terminal domain as an attractive therapeutic target. a Slice through cryo-tomographic volume of an individual mature capsid. Red arrows highlight CA pentamers. b The tomographic density matches the shape and size of the capsid, shown by the overlay of densities from the segmented capsid and the fullerene model (yellow). c Final molecular dynamics equilibrated all-atom capsid model. Images kindly provided by Peijun Zhang Univ. of Pittsburgh. Adapted from [22]. EMDB Accession codes: EMD-5582, EMD-5639; Fitted PDB IDs: 3J34, 3J4F, 3J3Y

Mentions: In the same publication the structure of the mature viral capsid was also determined. The HIV viral capsid forms a fullerene cone comprised of pentameric and hexameric capsid proteins. A fullerene cone requires 12 pentamers inserted into a hexagonal lattice to close the ovoid. However, there are several possible positions where pentamers can be inserted into the hexagonal lattice allowing for formation of the cone structure. This results in multiple forms of fullerene cones. Just as predicted by Euclidian geometry of fullerenes, HIV viral capsids also visually exhibit this expected variation in shape and geometry [24]. Since the HIV capsid it is a highly variable, pleomorphic structure and is therefore intractable to structural techniques that require averaging of structurally homogeneous particles. However CET is ideally suited to study such structures. CET is analogous to a CT scan, where a scanner rotates around a specimen (i.e. a person), collecting images at each tilt allowing for generation of a 3D reconstruction of the specimen. In CET, instead of tilting the microscope, the specimen is tilted within the column of the electron microscope [4, 25–27]. With this method 3D tomographic volumes of individual mature capsids were generated. Combining a lower resolution tomographic volume as a constraint, the structures of pentamers, hexamers, and novel contact sites determined by tubular analysis, allowed for creation of an atomic model of the entire HIV capsid and subsequently a whole atom molecular dynamic simulation of the entire capid as well (Fig. 5) (EMDB Accession codes: EMD-5582, EMD-5639; Fitted PDB IDs: 3J34, 3J4F, 3J3Y). To date it is the largest molecular dynamics simulation on a protein and was truly a heroic effort. More importantly the model highlighted the threefold capsid protein C-terminal domain as an attractive therapeutic target and provided extremely useful biochemical information.Fig. 5


Towards an integrative structural biology approach: combining Cryo-TEM, X-ray crystallography, and NMR.

Lengyel J, Hnath E, Storms M, Wohlfarth T - J. Struct. Funct. Genomics (2014)

Using cryo-tomographic analysis of individual, native “fullerene” cone HIV-1 capsid, combined with the high resolution Cryo-TEM hexamer structures an all-atom molecular dynamics HIV-1 capsid model was created. This model highlights the threefold capsid protein C-terminal domain as an attractive therapeutic target. a Slice through cryo-tomographic volume of an individual mature capsid. Red arrows highlight CA pentamers. b The tomographic density matches the shape and size of the capsid, shown by the overlay of densities from the segmented capsid and the fullerene model (yellow). c Final molecular dynamics equilibrated all-atom capsid model. Images kindly provided by Peijun Zhang Univ. of Pittsburgh. Adapted from [22]. EMDB Accession codes: EMD-5582, EMD-5639; Fitted PDB IDs: 3J34, 3J4F, 3J3Y
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125826&req=5

Fig5: Using cryo-tomographic analysis of individual, native “fullerene” cone HIV-1 capsid, combined with the high resolution Cryo-TEM hexamer structures an all-atom molecular dynamics HIV-1 capsid model was created. This model highlights the threefold capsid protein C-terminal domain as an attractive therapeutic target. a Slice through cryo-tomographic volume of an individual mature capsid. Red arrows highlight CA pentamers. b The tomographic density matches the shape and size of the capsid, shown by the overlay of densities from the segmented capsid and the fullerene model (yellow). c Final molecular dynamics equilibrated all-atom capsid model. Images kindly provided by Peijun Zhang Univ. of Pittsburgh. Adapted from [22]. EMDB Accession codes: EMD-5582, EMD-5639; Fitted PDB IDs: 3J34, 3J4F, 3J3Y
Mentions: In the same publication the structure of the mature viral capsid was also determined. The HIV viral capsid forms a fullerene cone comprised of pentameric and hexameric capsid proteins. A fullerene cone requires 12 pentamers inserted into a hexagonal lattice to close the ovoid. However, there are several possible positions where pentamers can be inserted into the hexagonal lattice allowing for formation of the cone structure. This results in multiple forms of fullerene cones. Just as predicted by Euclidian geometry of fullerenes, HIV viral capsids also visually exhibit this expected variation in shape and geometry [24]. Since the HIV capsid it is a highly variable, pleomorphic structure and is therefore intractable to structural techniques that require averaging of structurally homogeneous particles. However CET is ideally suited to study such structures. CET is analogous to a CT scan, where a scanner rotates around a specimen (i.e. a person), collecting images at each tilt allowing for generation of a 3D reconstruction of the specimen. In CET, instead of tilting the microscope, the specimen is tilted within the column of the electron microscope [4, 25–27]. With this method 3D tomographic volumes of individual mature capsids were generated. Combining a lower resolution tomographic volume as a constraint, the structures of pentamers, hexamers, and novel contact sites determined by tubular analysis, allowed for creation of an atomic model of the entire HIV capsid and subsequently a whole atom molecular dynamic simulation of the entire capid as well (Fig. 5) (EMDB Accession codes: EMD-5582, EMD-5639; Fitted PDB IDs: 3J34, 3J4F, 3J3Y). To date it is the largest molecular dynamics simulation on a protein and was truly a heroic effort. More importantly the model highlighted the threefold capsid protein C-terminal domain as an attractive therapeutic target and provided extremely useful biochemical information.Fig. 5

Bottom Line: In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques.Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable.Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.

View Article: PubMed Central - PubMed

Affiliation: FEI Company, 5350 N.E. Dawson Creek Drive, Hillsboro, OR, 97124, USA, Jeffrey.lengyel@fei.com.

ABSTRACT
Cryo-transmission electron microscopy (Cryo-TEM) and particularly single particle analysis is rapidly becoming the premier method for determining the three-dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques. While Cryo-TEM was once thought of as a low resolution structural technique, the method is currently capable of generating nearly atomic resolution structures on a routine basis. Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable. Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.

Show MeSH
Related in: MedlinePlus