Limits...
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.

Show MeSH

Related in: MedlinePlus

Use of time-resolved Cryo-TEM to visualize ribosome dynamics and tRNA movement. Furthermore, using the ratio of particles residing in each state, the entire thermodynamic dynamic landscape of the transition between states was determined. This is the first example of how using Cryo-TEM structural information to calculate thermodynamic parameters. Image kindly provided by Niels Fischer and Holger Stark, MPI Gottingen. Adapted from [19]. EMDB Accession codes: EMD-1716, EMD-1717, EMD-1718, EMD-1719, EMD-1720, EMD-1721, EMD-1722, EMD-1723, EMD-1724, EMD-1725, EMD-1726, EMD-1727; Fitted PDB IDs: 3J4V, 3J52, 3J4Z, 3J50, 3J4Y, 3J51, 3J53, 3J54, 3J57, 3J58, 3J59, 3J5A, 3J5B, 3J5C, 3J5H, 3J5J, 3J5J, 3J5K
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4125826&req=5

Fig3: Use of time-resolved Cryo-TEM to visualize ribosome dynamics and tRNA movement. Furthermore, using the ratio of particles residing in each state, the entire thermodynamic dynamic landscape of the transition between states was determined. This is the first example of how using Cryo-TEM structural information to calculate thermodynamic parameters. Image kindly provided by Niels Fischer and Holger Stark, MPI Gottingen. Adapted from [19]. EMDB Accession codes: EMD-1716, EMD-1717, EMD-1718, EMD-1719, EMD-1720, EMD-1721, EMD-1722, EMD-1723, EMD-1724, EMD-1725, EMD-1726, EMD-1727; Fitted PDB IDs: 3J4V, 3J52, 3J4Z, 3J50, 3J4Y, 3J51, 3J53, 3J54, 3J57, 3J58, 3J59, 3J5A, 3J5B, 3J5C, 3J5H, 3J5J, 3J5J, 3J5K

Mentions: Not only has Cryo-TEM been able to generate very high resolution reconstructions of the ribosome, it has proved invaluable to visualizing dramatic structural changes that occur during RNA translation. Using “4D” or time resolved Cryo-TEM, Fischer et al. [19] have been able to reconstruct numerous intermediate structural states of ribosome within a single dataset using advanced image processing algorithms (Fig. 3). Combining these structural intermediates resulted in a movie of the process of translation. Additionally, by determining the occupancy of each conformational state it was possible to calculate the entire thermodynamic landscape through the whole dynamic process. In total Cryo-TEM has resulted in numerous insights into ribosome biology. For instance Hashem et al. [20] have been able to show how viral messenger RNA can displace eukaryotic initiation factors in order to promote translation of viral mRNA (EMDB Accession codes: EMD-2450, EMD-2451; Fitted PDB ID: 4C4Q).Fig. 3


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)

Use of time-resolved Cryo-TEM to visualize ribosome dynamics and tRNA movement. Furthermore, using the ratio of particles residing in each state, the entire thermodynamic dynamic landscape of the transition between states was determined. This is the first example of how using Cryo-TEM structural information to calculate thermodynamic parameters. Image kindly provided by Niels Fischer and Holger Stark, MPI Gottingen. Adapted from [19]. EMDB Accession codes: EMD-1716, EMD-1717, EMD-1718, EMD-1719, EMD-1720, EMD-1721, EMD-1722, EMD-1723, EMD-1724, EMD-1725, EMD-1726, EMD-1727; Fitted PDB IDs: 3J4V, 3J52, 3J4Z, 3J50, 3J4Y, 3J51, 3J53, 3J54, 3J57, 3J58, 3J59, 3J5A, 3J5B, 3J5C, 3J5H, 3J5J, 3J5J, 3J5K
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Use of time-resolved Cryo-TEM to visualize ribosome dynamics and tRNA movement. Furthermore, using the ratio of particles residing in each state, the entire thermodynamic dynamic landscape of the transition between states was determined. This is the first example of how using Cryo-TEM structural information to calculate thermodynamic parameters. Image kindly provided by Niels Fischer and Holger Stark, MPI Gottingen. Adapted from [19]. EMDB Accession codes: EMD-1716, EMD-1717, EMD-1718, EMD-1719, EMD-1720, EMD-1721, EMD-1722, EMD-1723, EMD-1724, EMD-1725, EMD-1726, EMD-1727; Fitted PDB IDs: 3J4V, 3J52, 3J4Z, 3J50, 3J4Y, 3J51, 3J53, 3J54, 3J57, 3J58, 3J59, 3J5A, 3J5B, 3J5C, 3J5H, 3J5J, 3J5J, 3J5K
Mentions: Not only has Cryo-TEM been able to generate very high resolution reconstructions of the ribosome, it has proved invaluable to visualizing dramatic structural changes that occur during RNA translation. Using “4D” or time resolved Cryo-TEM, Fischer et al. [19] have been able to reconstruct numerous intermediate structural states of ribosome within a single dataset using advanced image processing algorithms (Fig. 3). Combining these structural intermediates resulted in a movie of the process of translation. Additionally, by determining the occupancy of each conformational state it was possible to calculate the entire thermodynamic landscape through the whole dynamic process. In total Cryo-TEM has resulted in numerous insights into ribosome biology. For instance Hashem et al. [20] have been able to show how viral messenger RNA can displace eukaryotic initiation factors in order to promote translation of viral mRNA (EMDB Accession codes: EMD-2450, EMD-2451; Fitted PDB ID: 4C4Q).Fig. 3

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