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Resolution Assessment in CryoEM

Resolution Assessment in CryoEM. Kristin Shingler Friday, August 2 nd , 2013. What is Resolution?. In short, how close two objects can be without appearing as one. Atomic resolution is achieved in x-ray crystallography.

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Resolution Assessment in CryoEM

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  1. Resolution Assessment in CryoEM Kristin Shingler Friday, August 2nd, 2013

  2. What is Resolution? • In short, how close two objects can be without appearing as one. • Atomic resolution is achieved in x-ray crystallography. • Typically, cryoEM generates sub-nanometer resolution, but recent reconstructions have approached atomic resolution.

  3. Semliki Forest Virus 1986 3.2 nanometerresolution Vogel et al.,1986 Nature

  4. Sub-nanometer Resolution Hepatitis B Capsid, 9.1ÅResolution Conway et al., 1997 Science

  5. Near-Atomic Resolution Epsilon 15 Bacteriophage, 4.5ÅResolution Jiang et al., 2008 Nature

  6. Atomic Resolution Human Adenovirus 5, 3.6ÅResolution Liu et al., 2010 Science

  7. What does resolution really mean? Zhou, Current Opinions in Structural Biology, 2008

  8. What does resolution really mean? Baker et al., Methods in Enzymology, 2010

  9. Resolution Assessment in X-ray Crystallography • Determined by how closely the crystal lattice can be packed, while still maintaining diffraction • Various statistical measurements correlate the resolution Modified Figure 5 from Wlodawer et al. 2007

  10. 3D Reconstruction Work Flow Baker, Olson, and Fuller 1999 Microbiology and Molecular Biology Reviews

  11. Fourier Shell Correlation F1 – Structure Factor for Map #1 F2 – Structure Factor for Map #2 ri – individual voxel element r – radius “measures the normliazed cross-correlation coefficient between two 3D volumes as a function of spatial frequency” - van Heel

  12. Conventional Resolution Estimation versus Gold Standard Conventional Gold Standard Model Model Model ½ Data Map ½ Data Map Even Map Odd Map FSC and Resolution Estimate FSC and Resolution Estimate

  13. Conventional Resolution Estimation versus Gold Standard Scheres and Chene, 2012 Nature Methods

  14. Why this paper? • Elife is an open access journal • It provides readers with both the reviewers comments AND the author’s response to reviewers • An excellent example of high quality work showing the disparity between conventional and gold standard resolution assessment

  15. Timeline of Events • The authors first reported the resolution of their map to be 4.6Å by using conventional resolution assessment • This method was challenged by reviewers • The authors reassessed resolution using the gold standard method and found the map was 5.3Å resolution

  16. Reviewers Comment Sun et al.,2013Elife

  17. Author’s Response Sun et al.,2013Elife

  18. Chikungunya Virus Structure Sun et al.,2013Elife

  19. CryoEM Density Fitting Sun et al.,2013Elife

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