1. Resolution Assessment in CryoEM Kristin Shingler Friday, August 2 nd, 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 nanometer r esolution Vogel et al., 1986 Nature

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

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

6. Atomic Resolution Human Adenovirus 5, 3.6Å Resolution 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 Modified Figure 5 from Wlodawer et al. 2007 Determined by how closely the crystal lattice can be packed, while still maintaining diffraction Various statistical measurements correlate the resolution

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

11. Fourier Shell Correlation F 1 – Structure Factor for Map #1 F 2 – Structure Factor for Map #2 r i – 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 ConventionalGold Standard Model Even Map Odd Map FSC and Resolution Estimate Model ½ Data Map ½ Data Map FSC and Resolution Estimate

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

15. 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

16. 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

17. Reviewers Comment Sun et al.,2013 Elife

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

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

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