stanford-slac cryo-electron microscopy - dealing with … · 2020. 8. 12. · fourier transform...

Dealing with Resolution Anisotropy andConsiderations for High Resolution Structure

Determination

Dmitry LyumkisSalk Institute for Biological Studies

American Crystallographic Association, cryo-EM Workshop, August 11, 2020

• Preferred orientation — the origin of resolution anisotropy

Outline

• Resolution anisotropy in cryo-EM reconstructions (caused

by preferred orientation) and its evaluation with 3D FSCs

• Combatting anisotropy using tilted data collection strategies

• The (unexpected) effect of sampling (in)homogeneity on

global resolution

• Evaluating sampling (in)homogeneity using the SCF criterion

• Considerations for high-resolution

Origin of resolution anisotropy in cryo-EM:The idealized behavior of particles on a grid

Noble, A. J., et al. (2018). Routine single particle CryoEM sample and grid characterization bytomography. eLife, 7, 32.


Origin of resolution anisotropy in cryo-EM:The reality … Particles adhere at the air-water interface

Origin of resolution anisotropy in cryo-EM:The reality … for virtually all samples



Outline





global resolution



90°

Interaction at the air-water interface results in:“preferred particle orientation” — Hemagglutinin (HA) trimer

Tan, Y. Z. et al. (2017). Nature Methods, 14(8), 793–796.

Interaction at the air-water interface results in:smearing and elongation along Z-axis (loss of Z resolution)


beamdirection

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Fourier Shell Correlation (FSC): a quantitative way toevaluate resolution in cryo-EM

● Calculate the FSC between two half maps as afunction of spatial frequency over the entireFourier shell

Harauz, G., & van Heel, M. (1986). Exact filters for general geometry 3-dimensionalreconstruction. Optik, 73(146), 146–156.

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FSC 0.143


● Calculate the FSC between two half maps as afunction of the direction (and looking atcross-correlations over a local cone of dataand not the entire shell)

3D FSCs: a quantitative way to evaluate directional resolutionand anisotropy in single-particle cryo-EM

Tan, Y. Z. …, Lyumkis D. (2017). Nature Methods, 14(8), 793–796.


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FSC at View #1




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FSC at View #2




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Conical FSCsAcross Entire

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Conical FSCsAcross Entire

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3D FSC



Evaluating resolution anisotropy: the 3D FSC

Tan et al. (2017). Nat Methods, 14(8), 793-796. Aiyer et al. in press. MiMB.

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Implementation of 3D FSCs in Chimera

Aiyer et al. in press. MiMB.

Web-server for remote 3D FSC processing:https://3dfsc.salk.edu

www.github.com/LyumkisLab/3DFSC

http://www.github.com/LyumkisLab/3DFSC


Outline





global resolution



Tilting the grid in the microscope helps to alleviateresolution anisotropy caused by preferred specimen

orientation

Lyumkis, D. (2019). JBC, 294(13), 5181–5197.

Tan et al. (2017). Nat Methods, 14(8), 793-796.

Why do tilts work?Filling in more Fourier space density (better Fourier sampling)

reconstruct

reconstruct

Tilting ameliorates resolution anisotropy and improves maps

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Anisotropic reconstructionuntilted data

(more) isotropic reconstructiontilted data

Passos, Li et al. (2020). Science

Tilting ameliorates resolution anisotropy and improves mapsHIV-1 intasome complexes (from Thursday ACA session)

Examples of tilted data collection strategies in the literature

Zhang, Y., … Shi, Y. (2020). bioRxiv, 9,2020.03.27.009381

~100 MDa Nuclear Pore Complex

Herzik, M. A., Wu, M., & Lander, G. C. (2019).Nature Communications, 10(1), 1–9.

~43 kDa Protein Kinase A

Possible limitations associated with tilted datacollection strategies

1. Defocus gradient across the micrograph

2. More beam-induced movement

3. Thicker ice

Compensating for limitations of tilts usingcomputational image processing: AAV

Working hypothesis: most of the problems associated with tilts couldbe ameliorated using computational strategies

0°st

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Outline





global resolution



75K particles

Passos, D. O., & Lyumkis, D. (2015). Journal of Structural Biology, 192(2), 235–244.

High-resolution dataset, with multiple “preferredorientations” for characterizing the effects of sampling

75K particles 10K particles

10K particlesorientation 1



Dataset could be subdivided into four groups by orientation

Reconstructions differing only in orientation distributionshave distinct SSNR profiles


Outline





global resolution



Dataset signal power /noise power at different

spatial frequencies

Average numberof times lattice

point k is sampled

Geometricalfactor

Per-particle SSNR

Heymann, J. B., IUCr. (2019). Single-particle reconstruction statistics: a diagnostic tool in solving biomolecularstructures by cryo-EM. Acta Crystallographica. Section F, Structural Biology and Crystallization Communications,

75(1), 33–44.

Decoupling sampling with noise variance

Derivation decouples a sampling factor from the per-particle SSNR

Previously …

Baldwin, P. R., & Lyumkis, D. (2019). Progress in Biophysics and Molecular Biology.http://doi.org/10.1016/j.pbiomolbio.2019.09.002

Evaluating SCF behavior using different Fourier samplingschemes

Decrement of the SCF for side-like views: (fully sampled)

Decrement of the SCF for top-like views:(Incompletely sampled)

45-degree cone,10% sprinkles




Uniform

Knowing the SCF can predict the attenuation of the SSNR:side-like distributions, varying phi-angle modulation

Knowing the SCF can predict the attenuation of the SSNR:top-like distributions, varying cone size with 3% “sprinkles”

GUI for calculating SCF values:Example with top-like distribution, untilted and tilted

Baldwin, P. R., & Lyumkis, D. (2020).bioRxiv, 3(1), 2020.06.08.140863.

www.github.com/LyumkisLab/SamplingGui

Baldwin, P. R., & Lyumkis, D. (2020).PBMB, Jul 6:S0079-6107(20)30060-2

SCF* = 0.02

SCF* = 0.49

http://www.github.com/LyumkisLab/SamplingGui


Outline





global resolution



Considerations for optimizing cryo-EM resolution• Microscope (e.g. JEOL, TFS, Krios G3/G4, Arctica, etc) microscope settings (e.g. KV, aperture

size), microscope alignment (coma-free, parallel illumination)• Ancillary hardware: cold or warm FEG, energy filter, aberration correctors, phase plates• Detector and detector DQE: Falcon4, Falcon3, K3, K2, CCD, etc• Detector settings: counting vs. super-resolution, dose rate, frame rate• Imaging and data collection: magnification (and pixel size), defocus range, image shift or stage

position, speed vs accuracy (without compromising quality), hole selection, with or w/o stage tilt

• Grid / data collection quality: thick/thin ice, how much drift, sample dispersity, substrate support,grid type

• Image processing:◦ Software (Relion, CryoSparc, CisTEM, Warp/M, EMAN, etc)◦ Initial motion correction and CTF estimation (global, patch-based, per-particle)◦ Classification strategy◦ Post-processing (CTF refinement, particle polishing [movement, B-factor weighting], Ewald

correction, density modification)

• Fourier space sampling• Sample !!!!!!!!!!!!!!!!!!!!!!!!!

Nakane, T., …, Scheres, S. (2020). Single-particle cryo-EM at atomic resolution.bioRxiv, 2020.05.22.110189.

Considerations for optimizing cryo-EM resolution:1.2 Å apoferritin (cold FEG)

Yip, K. M., Fischer, N., Paknia, E., Chari, A., & Stark, H. (2020). Breaking the next Cryo-EMresolution barrier – Atomic resolution determination of proteins! bioRxiv, 645,

2020.05.21.106740.

Considerations for optimizing cryo-EM resolution:1.2 Å apoferritin (warm FEG)

• Titan Krios (Schottky FEG)• Voltage: 300• Monochromator + Cs corrector• Falcon 3 detector• 0.492 Å/pix• Electron dose (e-/Å^2): 50• Software: Relion• Number of particles: 1,090,676• Resolution: 1.25 Å

Considerations for optimizing cryo-EM resolution:1.3 Å apoferritin (cold FEG)

Tegunov, D., Xue, L., Dienemann, C., Cramer, P., & Mahamid, J. (2020). Multi-particle cryo-EM refinement with Mvisualizes ribosome-antibiotic complex at 3.7 Å inside cells. bioRxiv, 44, 2020.06.05.136341.

• cryoARM300 (JEOL)• Voltage: 300• Cold FEG• K2 detector• In-column omega filter• 0.49 Å/pix• Electron dose (e-/Å^2): 88• Software: M• Number of particles: 120,295• Resolution: 1.34 Å

Dimitry TegunovReprocessed dataset in M

EMPIAR-10248

Keiichi NambaData collection (Kato et al, MIcroscopy

and Microanalysis, 2019)

Rado DanevYanagisawa-san, Masahide

Kikkawa

Considerations for optimizing cryo-EM resolution:1.3 Å apoferritin (warm FEG)

Try to understand your limitationsFirst 2 e-/Å2

Tan, Y. Z., Aiyer, S., et al. (2018). Nature Communications, 9(1), 3628–11.

Exposure Dependency of the Mean Intensityof the Unmerged Integrated Reflections

Hattne, J., Shi, D., et al. (2018). Analysis of Global and Site-Specific Radiation Damage inCryo-EM, 26(5), 759–766.e4.

• Resolution anisotropy originates from preferred orientation and

inhomogeneity in Fourier sampling

• 3D FSCs provide quantitative measurements of resolution

anisotropy

• Tilts help to overcome resolution anisotropy

• Inhomogeneity in Fourier sampling also causes attenuation of

global resolution: incomplete sampling worse than

inhomogeneous (but complete) sampling

• The Sampling Compensation Factor (SCF) estimates the

amount of global resolution attenuation

• Many factors to optimize for high-resolution … toward sub-Å

reconstructions!

Summary

Acknowledgements and Support

Computationalsupport

Phil BaldwinJC Ducom

Funding

Collaborators

Niko GrigorieffBen Himes

Mavis Agbanjie-McKenna

Robert McKenna

Cryo-EM supportBill Anderson

Scripps/SalkConsortium

Jamie WilliamsonTravis BerggrenMartin Hetzer

Leona M. and Harry B. HelmsleyCharitable Trust Grant

Phil BaldwinYong Zi Tan Sriram Aiyer

stanford-slac cryo-electron microscopy - dealing with … · 2020. 8. 12. · fourier transform...

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