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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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a 3.3 Å structure of a non-enveloped icosahedral virus using Cryo-TEM. b Image of a selected region were individual side chains are clearly visible in the Cryo-TEM reconstruction validating the high resolution of the reconstruction. Images kindly provided by Z. Hong Zhou Electron Imaging Center for Nanomachines (EICN) CNSI and Department of Microbiology, Immun & Mol. Genetics, UCLA, and adapted from [9]. EMDB Accession code: EMD-5160; Fitted PDB ID: 3IYL
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Fig1: a 3.3 Å structure of a non-enveloped icosahedral virus using Cryo-TEM. b Image of a selected region were individual side chains are clearly visible in the Cryo-TEM reconstruction validating the high resolution of the reconstruction. Images kindly provided by Z. Hong Zhou Electron Imaging Center for Nanomachines (EICN) CNSI and Department of Microbiology, Immun & Mol. Genetics, UCLA, and adapted from [9]. EMDB Accession code: EMD-5160; Fitted PDB ID: 3IYL

Mentions: Classically single particle analysis, particularly when applied to Cryo-TEM studies, has been thought of as a very low resolution (~30 Å resolution) structural technique that can be applied to only very rigid, megadalton sized protein complexes; such as the ribosome, or highly symmetric icosahedral viruses. While large protein complexes and viruses are still being studied, Cryo-TEM has undergone a revolution, aided by advancements in sample preparation, electron microscope technology, automation in data collection, detector technology, and image processing [2–5]. Taken as a whole, these advancements have allowed Cryo-TEM researchers to determine protein structures at atomic resolution and addressed biological research questions previously thought unattainable by the method. For instance in 2009 Zhang et al. [9] published the seminal atomic resolution 3.3 Å aquareovirus structure in the journal Cell where individual side chains were clearly resolved (Fig. 1) (EMDB Accession code: EMD-5160; Fitted PDB ID: 3IYL). Due to the extremely high resolution obtained in the structure, autolytic cleavages site were visualized which revealed a novel priming mechanism for cell entry for non-enveloped viruses. The same year Liu et al. [10] published a 3.6 Å structure of adenovirus in the journal Science (EMDB Accession code: EMD-5172; Fitted PDB ID: 3IYN). In the same issue, an X-ray crystal structure of adenovirus at 3.5 Å was also published [11] (PDB ID: 1VSZ). Steven Harrison (Harvard) wrote in a commentary about the two structures, stating that while the X-ray structure was at higher resolution, the Cryo-TEM structure actually showed substantially better details on a more difficult and more biologically relevant type of the virus [12]. Cryo-TEM had unequivocally bested X-ray crystallography when applied to viruses and is currently considered the premier method for icosahedral virus structure determination. This is further echoed in a recent publication (confessions of an icosahedral virus crystallographer) by structural virologist, Jack Johnson (Scripps Research Institute), where he details his methodological change from using X-ray crystallography to study virus structure to solely using Cryo-TEM [13].Fig. 1


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)

a 3.3 Å structure of a non-enveloped icosahedral virus using Cryo-TEM. b Image of a selected region were individual side chains are clearly visible in the Cryo-TEM reconstruction validating the high resolution of the reconstruction. Images kindly provided by Z. Hong Zhou Electron Imaging Center for Nanomachines (EICN) CNSI and Department of Microbiology, Immun & Mol. Genetics, UCLA, and adapted from [9]. EMDB Accession code: EMD-5160; Fitted PDB ID: 3IYL
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: a 3.3 Å structure of a non-enveloped icosahedral virus using Cryo-TEM. b Image of a selected region were individual side chains are clearly visible in the Cryo-TEM reconstruction validating the high resolution of the reconstruction. Images kindly provided by Z. Hong Zhou Electron Imaging Center for Nanomachines (EICN) CNSI and Department of Microbiology, Immun & Mol. Genetics, UCLA, and adapted from [9]. EMDB Accession code: EMD-5160; Fitted PDB ID: 3IYL
Mentions: Classically single particle analysis, particularly when applied to Cryo-TEM studies, has been thought of as a very low resolution (~30 Å resolution) structural technique that can be applied to only very rigid, megadalton sized protein complexes; such as the ribosome, or highly symmetric icosahedral viruses. While large protein complexes and viruses are still being studied, Cryo-TEM has undergone a revolution, aided by advancements in sample preparation, electron microscope technology, automation in data collection, detector technology, and image processing [2–5]. Taken as a whole, these advancements have allowed Cryo-TEM researchers to determine protein structures at atomic resolution and addressed biological research questions previously thought unattainable by the method. For instance in 2009 Zhang et al. [9] published the seminal atomic resolution 3.3 Å aquareovirus structure in the journal Cell where individual side chains were clearly resolved (Fig. 1) (EMDB Accession code: EMD-5160; Fitted PDB ID: 3IYL). Due to the extremely high resolution obtained in the structure, autolytic cleavages site were visualized which revealed a novel priming mechanism for cell entry for non-enveloped viruses. The same year Liu et al. [10] published a 3.6 Å structure of adenovirus in the journal Science (EMDB Accession code: EMD-5172; Fitted PDB ID: 3IYN). In the same issue, an X-ray crystal structure of adenovirus at 3.5 Å was also published [11] (PDB ID: 1VSZ). Steven Harrison (Harvard) wrote in a commentary about the two structures, stating that while the X-ray structure was at higher resolution, the Cryo-TEM structure actually showed substantially better details on a more difficult and more biologically relevant type of the virus [12]. Cryo-TEM had unequivocally bested X-ray crystallography when applied to viruses and is currently considered the premier method for icosahedral virus structure determination. This is further echoed in a recent publication (confessions of an icosahedral virus crystallographer) by structural virologist, Jack Johnson (Scripps Research Institute), where he details his methodological change from using X-ray crystallography to study virus structure to solely using Cryo-TEM [13].Fig. 1

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