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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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9 Å three-dimensional reconstruction of soluble gp140 HIV envelope glycoprotein trimers bound to three copies of the Fab fragment from 17b, a neutralizing antibody whose binding mimics that of the co-receptor. The structure revealed the presence of a previously unknown “activated” intermediate state, where three buried helices become exposed and potentially accessible to binding by entry inhibitors. a Top view of the trimer and b Side view of the trimer. Images kindly provided by Sriram Subramaniam, Lab of Cell Biology, National Cancer Institute, National Institutes of Health. Adapted from [16]. EMDB Accession code: EMD-5462
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Fig2: 9 Å three-dimensional reconstruction of soluble gp140 HIV envelope glycoprotein trimers bound to three copies of the Fab fragment from 17b, a neutralizing antibody whose binding mimics that of the co-receptor. The structure revealed the presence of a previously unknown “activated” intermediate state, where three buried helices become exposed and potentially accessible to binding by entry inhibitors. a Top view of the trimer and b Side view of the trimer. Images kindly provided by Sriram Subramaniam, Lab of Cell Biology, National Cancer Institute, National Institutes of Health. Adapted from [16]. EMDB Accession code: EMD-5462

Mentions: Cryo-TEM is also rapidly being applied to smaller (sub-megadalton) protein complexes. For instance Cryo-TEM has been proven the superior method for studying the trimeric HIV surface spike and has been instrumental in proving that the spike can undergo dramatic conformational changes upon binding to the target host cell proteins [14–16] (Fig. 2) (EMDB Accession codes: EMD-5462; Fitted PDB ID: 3IYN). Visualization of these conformational states could prove critical in future drug design. Currently, numerous research groups are now applying Cryo-TEM methods to sub 500 kDa sized proteins complexes, and recently Wu et al. [17] published a sub-nanometer structure of HIV integrase bound to Fabs resulting in a total molecular weight of 110 kDa (EMDB Accession code: EMD-5294).Fig. 2


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)

9 Å three-dimensional reconstruction of soluble gp140 HIV envelope glycoprotein trimers bound to three copies of the Fab fragment from 17b, a neutralizing antibody whose binding mimics that of the co-receptor. The structure revealed the presence of a previously unknown “activated” intermediate state, where three buried helices become exposed and potentially accessible to binding by entry inhibitors. a Top view of the trimer and b Side view of the trimer. Images kindly provided by Sriram Subramaniam, Lab of Cell Biology, National Cancer Institute, National Institutes of Health. Adapted from [16]. EMDB Accession code: EMD-5462
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: 9 Å three-dimensional reconstruction of soluble gp140 HIV envelope glycoprotein trimers bound to three copies of the Fab fragment from 17b, a neutralizing antibody whose binding mimics that of the co-receptor. The structure revealed the presence of a previously unknown “activated” intermediate state, where three buried helices become exposed and potentially accessible to binding by entry inhibitors. a Top view of the trimer and b Side view of the trimer. Images kindly provided by Sriram Subramaniam, Lab of Cell Biology, National Cancer Institute, National Institutes of Health. Adapted from [16]. EMDB Accession code: EMD-5462
Mentions: Cryo-TEM is also rapidly being applied to smaller (sub-megadalton) protein complexes. For instance Cryo-TEM has been proven the superior method for studying the trimeric HIV surface spike and has been instrumental in proving that the spike can undergo dramatic conformational changes upon binding to the target host cell proteins [14–16] (Fig. 2) (EMDB Accession codes: EMD-5462; Fitted PDB ID: 3IYN). Visualization of these conformational states could prove critical in future drug design. Currently, numerous research groups are now applying Cryo-TEM methods to sub 500 kDa sized proteins complexes, and recently Wu et al. [17] published a sub-nanometer structure of HIV integrase bound to Fabs resulting in a total molecular weight of 110 kDa (EMDB Accession code: EMD-5294).Fig. 2

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