Resolution Assessment in CryoEM

Kristin ShinglerFriday, August 2nd, 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, cryoEM 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

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

3D Reconstruction Work Flow

Baker, Olson, and Fuller 1999 Microbiology and Molecular Biology Reviews

Fourier Shell Correlation

F1 – Structure Factor for Map #1F2 – Structure Factor for Map #2ri – individual voxel elementr – radius

“measures the normliazed cross-correlation coefficient between two 3D volumes as a function of spatial frequency” - van Heel

Conventional Resolution Estimation versus Gold Standard

Conventional Gold Standard

Model

EvenMap

OddMap

FSC and Resolution Estimate

Model Model

½DataMap

½DataMap

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

CryoEM Density Fitting

Sun et al.,2013 Elife