Resolution Assessment in Cryo EM Kristin Shingler Friday

Resolution Assessment in Cryo. EM 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. • Typically, cryo. EM generates sub-nanometer resolution, but recent reconstructions have approached atomic resolution.

Semliki Forest Virus 1986 3. 2 nanometer resolution Vogel et al. , 1986 Nature

Sub-nanometer Resolution Hepatitis B Capsid, 9. 1Å Resolution Conway et al. , 1997 Science

Near-Atomic Resolution Epsilon 15 Bacteriophage, 4. 5Å Resolution Jiang et al. , 2008 Nature

Atomic Resolution Human Adenovirus 5, 3. 6Å Resolution Liu et al. , 2010 Science

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

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

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

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

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

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

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

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

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

Reviewers Comment Sun et al. , 2013 Elife

Author’s Response Sun et al. , 2013 Elife

Chikungunya Virus Structure Sun et al. , 2013 Elife

Cryo. EM Density Fitting Sun et al. , 2013 Elife