Electron tomography for SARS-CoV-2 100 nm False colour TEM image - - PowerPoint PPT Presentation

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Electron tomography for SARS-CoV-2 100 nm False colour TEM image - - PowerPoint PPT Presentation

Nov 29, 2022 464 likes •937 views

Electron tomography for SARS-CoV-2 100 nm False colour TEM image from NIAID, USA Alan Roseman -- University of Manchester FEI Polara 300 kV TEM in FLS, UoM. Nextstrain Alan Roseman -- University of Manchester Alan Roseman -- University of

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Alan Roseman -- University of Manchester

Electron tomography for SARS-CoV-2

False colour TEM image from NIAID, USA 100 nm

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FEI Polara 300 kV TEM in FLS, UoM.

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Nextstrain

Alan Roseman -- University of Manchester

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Alan Roseman -- University of Manchester

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Nextstrain

Alan Roseman -- University of Manchester

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Nextstrain

Alan Roseman -- University of Manchester

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Nextstrain

Alan Roseman -- University of Manchester

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Alan Roseman -- University of Manchester

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Model of virus and virus proteins

Alan Roseman -- University of Manchester

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TEM of SARS-CoV-2 (from NIAID)

Alan Roseman -- University of Manchester

100 nm

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TEM of SARS-CoV-2

Alan Roseman -- University of Manchester

TEM of SARS-CoV-2 (from NIAID)

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TEM of SARS-CoV-2

Alan Roseman -- University of Manchester TEM of SARS-CoV-2 (from NIAID)

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Spike density at 20 Å resoluPon

Alan Roseman -- University of Manchester

virus surface 25 nm

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Electron tomography for SARS-CoV-2

Alan Roseman -- University of Manchester

100 nm

TEM of SARS-CoV-2 (from NIAID)

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Electron tomography

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Sample preparaPon

  • H. Saibil

Alan Roseman -- University of Manchester

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Cryo EM

  • H. Saibil
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EM ET principles

  • Parallel, coherent, electron beam
  • Missing wedge
  • Defocus/height dependent contrast transfer funcPon
  • Dose damage
  • DeformaPon/flow of sample
  • (correcPons for these are applied in current

soVware, all would improve from local, spaPal,

  • pPmisaPon)

Alan Roseman -- University of Manchester

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Alan Roseman -- University of Manchester

MHV (betacoronavirus)

100 nm MHV cryoET, MHV is also a betacoronavirus

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Issues with biological ET

  • Low SNR
  • Missing wedge
  • Progressive sample damage by beam
  • Unstable sample
  • High resoluPon obtained by averaging of

idenPcal “moPfs” as sub-tomograms.

Alan Roseman -- University of Manchester

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Model virion with variable spikes

Alan Roseman -- University of Manchester

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Alan Roseman -- University of Manchester

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Alan Roseman -- University of Manchester

Single parPcle workflow

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100 nm

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Alan Roseman -- University of Manchester

Figure S3. Cryo-EM data processing workflow Cryo-EM structure of the 2019-nCoV spike in the prefusion conformaCon . Wrapp et al, Science 2020.

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Alan Roseman -- University of Manchester

Extended Data Figure 2 from: A.C. Walls, M.A. Tortorici, B.J. Bosch, B. Frenz, P.J.M. Ro_er, F. DiMaio, F.A. Rey, D. Veesler Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer Nature, 531 (2016), pp. 114-117 Scale bars: 573 Å (micrograph) and 44 Å (class averages)

CryoEM image of isolated coronavirus spike proteins

2D class averages

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WT cores WT fab labelled

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WT cores WT fab labelled 360 Å 500 Å

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Alan Roseman -- University of Manchester

Back to those spikes……

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Alan Roseman -- University of Manchester

ACE2

Adapted from: Vivian A. Scheuplein et al. J. Virol. 2015; doi:10.1128/JVI.03607-14

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Spike density at 20 Å resoluPon

Alan Roseman -- University of Manchester

The atomic models of the spike were determined by Walls et al (2020), using the cryoEM “single parPcle” method. Stabilised soluble spike proteins were produced in isolaPon, from mutated gene sequences, in lab grown cells. Structure, FuncCon, and AnCgenicity of the SARS-CoV-2 Spike Glycoprotein Alexandra C. Walls, Young-Jun Park, M. Alejandra Tortorici, Abigail Wall, Andrew T. McGuire, David Veesler

  • Cell. 2020 Apr 16;181(2):281-292.e6. doi: 10.1016/j.cell.2020.02.058. Epub 2020 Mar 9.
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Spike density at 20 Å resoluPon

Alan Roseman -- University of Manchester

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Spike density at 20 Å resoluPon

Alan Roseman -- University of Manchester

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Spike density at 20 Å resoluPon

Alan Roseman -- University of Manchester

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Spike density at 20 Å resoluPon

Alan Roseman -- University of Manchester

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Spike density at 20 Å resoluPon

Alan Roseman -- University of Manchester

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Spike density at 20 Å resoluPon

Alan Roseman -- University of Manchester

virus surface anPbody spikes

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Alan Roseman -- University of Manchester

ACE2

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Model

Alan Roseman -- University of Manchester

Main challenges:

All general ET sample and dose issues

  • menPoned. E.g.

Missing wedge Defocus/height dependent contrast transfer funcPon Dose damage, low signal DeformaPon/flow of sample

In addiHon:

MulPple overlapping moPfs. Unknown level of variability.

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FEI Polara 300 kV TEM in FLS, UoM.

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Helen Saibil

Viruses and the Development of QuanCtaCve Biological Electron Microscopy

Notes Rec. R. Soc. Lond. 2004. Medical Research Council - Laboratory of Molecular Biology Cambridge Jo Butler, Samantha Wynne, John Berriman

R.A. Crowther

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HIV trimer

Nature, Vol 455|4 September 2008|doi:10.1038/nature07159

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Test data and procedures to demonstrate steps and features to model for high resoluPon analysis

This raw cryoEM data has been processed to resolve the capsid protein moPf to 3.1Å resoluPon, using sub-tomogram averaging. It has all the features of a good tomography dataset. The moPf is hexameric, and is probably not very heterogeneous. The challenge for coronaviurs is that in addiPon to a missing wedge, dose damage, etc; it has mulPple overlapping moPfs (the spikes) with an unknown level of variabiliy. Databank reference for images: EMPIAR-10164 hjps://www.ebi.ac.uk/pdbe/emdb/empiar/entry/10164/ This HIV-1 capsid protein moPf has been resolved and analysed, and published 3 Pmes:

  • Pub1. Schur, F. K. M. et al. An atomic model of HIV-1 capsid-SP1 reveals structures regulaPng

assembly and maturaPon. Science 353, 506–508 (2016). 3.9Å, EMD-4015

  • Pub2. Turoňová, B., Schur, F. K. M., Wan, W. & Briggs, J. A. G. Efficient 3D-CTF correcPon for cryo-

electron tomography using NovaCTF improves subtomogram averaging resoluPon to 3.4Å. J. Struct.

  • Biol. 199, 187–195 (2017). 3.4Å, EMD-3782
  • Pub3. Benjamin A. Himes & Peijun Zhang. emClarity: soVware for high-resoluPon cryo-electron

tomography and subtomogram averaging .Nature Methods, volume 15, pages. 955–961 (2018). 3.1Å, EMD-8986

Alan Roseman -- University of Manchester