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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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Related in: MedlinePlus

Cryo-TEM reveals various conformational states of areolysin pore formation where a prepore state transitions to a functional toxin pore inserted in the target membrane (a). Combining X-ray crystallography, Cryo-TEM, and molecular dynamics modeling revealed a novel swirling membrane insertion mechanism to form the pore, allowing for an atomic resolution interpretation of the transition from monomer to prepore to the functional pore state (b). Images kindly provided by Matteo Dal Peraro, Biomolecular Modeling—LBM Institute of Bioengineering, School of Life Sciences École Polytechnique Fédérale de Lausanne—EPFL & Swiss Institute of Bioinformatics—SIB Adapted from [20]
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Fig4: Cryo-TEM reveals various conformational states of areolysin pore formation where a prepore state transitions to a functional toxin pore inserted in the target membrane (a). Combining X-ray crystallography, Cryo-TEM, and molecular dynamics modeling revealed a novel swirling membrane insertion mechanism to form the pore, allowing for an atomic resolution interpretation of the transition from monomer to prepore to the functional pore state (b). Images kindly provided by Matteo Dal Peraro, Biomolecular Modeling—LBM Institute of Bioengineering, School of Life Sciences École Polytechnique Fédérale de Lausanne—EPFL & Swiss Institute of Bioinformatics—SIB Adapted from [20]

Mentions: Single particle analysis has resulted in numerous biological insights, however as is the case with several other research methods, it is the combined use of various techniques that often result in the most significant scientific advancements, where the whole is greater than the sum of its individual parts. There is strong belief that hybrid methods are the future of structural biology, with Cryo-TEM being an essential method in these studies. In this context, two studies in particular, combining Cryo-TEM, X-ray crystallography, and molecular dynamics simulations have resulted in groundbreaking results. For instance, a recent publication combined Cryo-TEM structural studies of the areolysin pore with X-ray crystallography and molecular dynamics analysis [21]. Areolysin is a member of an important and widely distributed pore forming protein. There were several essential questions in the pore forming field, such as how areolysin like proteins oligomerize and form pores in the membrane. To address these questions Degiacomi et al., used Cryo-TEM single particle analysis to generate 3D reconstructions in three intermediate states ranging from a heptameric prepore state to a transition to the pore state (Fig. 4).Fig. 4


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)

Cryo-TEM reveals various conformational states of areolysin pore formation where a prepore state transitions to a functional toxin pore inserted in the target membrane (a). Combining X-ray crystallography, Cryo-TEM, and molecular dynamics modeling revealed a novel swirling membrane insertion mechanism to form the pore, allowing for an atomic resolution interpretation of the transition from monomer to prepore to the functional pore state (b). Images kindly provided by Matteo Dal Peraro, Biomolecular Modeling—LBM Institute of Bioengineering, School of Life Sciences École Polytechnique Fédérale de Lausanne—EPFL & Swiss Institute of Bioinformatics—SIB Adapted from [20]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125826&req=5

Fig4: Cryo-TEM reveals various conformational states of areolysin pore formation where a prepore state transitions to a functional toxin pore inserted in the target membrane (a). Combining X-ray crystallography, Cryo-TEM, and molecular dynamics modeling revealed a novel swirling membrane insertion mechanism to form the pore, allowing for an atomic resolution interpretation of the transition from monomer to prepore to the functional pore state (b). Images kindly provided by Matteo Dal Peraro, Biomolecular Modeling—LBM Institute of Bioengineering, School of Life Sciences École Polytechnique Fédérale de Lausanne—EPFL & Swiss Institute of Bioinformatics—SIB Adapted from [20]
Mentions: Single particle analysis has resulted in numerous biological insights, however as is the case with several other research methods, it is the combined use of various techniques that often result in the most significant scientific advancements, where the whole is greater than the sum of its individual parts. There is strong belief that hybrid methods are the future of structural biology, with Cryo-TEM being an essential method in these studies. In this context, two studies in particular, combining Cryo-TEM, X-ray crystallography, and molecular dynamics simulations have resulted in groundbreaking results. For instance, a recent publication combined Cryo-TEM structural studies of the areolysin pore with X-ray crystallography and molecular dynamics analysis [21]. Areolysin is a member of an important and widely distributed pore forming protein. There were several essential questions in the pore forming field, such as how areolysin like proteins oligomerize and form pores in the membrane. To address these questions Degiacomi et al., used Cryo-TEM single particle analysis to generate 3D reconstructions in three intermediate states ranging from a heptameric prepore state to a transition to the pore state (Fig. 4).Fig. 4

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