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High-resolution Cryo-EM Structure of the Trypanosoma brucei Ribosome: A Case Study

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Computational Methods for Three-Dimensional Microscopy Reconstruction

Part of the book series: Applied and Numerical Harmonic Analysis ((ANHA))

Abstract

Single-particle cryo-electron microscopy has the immense advantage over crystallography in being able to image frozen-hydrated biological complexes in their “native” state, in solution. For years the ribosome has been the benchmark sample for particles without symmetry. It has witnessed steady improvement in resolution from the very first single-particle 3D reconstruction to today’s reconstructions at near-atomic resolution. In this study, we describe the different steps of sample preparation, data collection, data processing, and modeling that led to the 5Å structure of the T. brucei ribosome [32]. A local resolution estimation demonstrates the extent to which resolution can be anisotropic and pinpoints regions of higher heterogeneity or structural flexibility. This study also shows an example of misuse of spatial frequency filters leading to overfitting of the data and the artifacts that can be observed in the resulting density map.

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Abbreviations

1D:

One-dimensional

2D:

Two-dimensional

3D:

Three-dimensional

Å:

Angstrom

β-me:

2-Mercaptoethanol

CCD:

Charge-coupled device

CMOS:

Complementary metal-oxide-semiconductor

cryo-EM:

cryo-Electron microscopy

CTF:

Contrast transfer function

e- :

Electron

EGTA:

Ethylene glycol tetraacetic acid

eIF1:

Eukaryotic Initiation Factor 1

EM:

Electron microscopy

EMDB:

Electron microscopy data bank

eEF2:

Eukaryotic elongation factor 2

ES:

Expansion segment

FEG:

Field emission gun

FSC:

Fourier shell correlation

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

kV:

Kilovolt

LSU:

Large ribosomal subunit

MDFF:

Molecular dynamics flexible fitting

MDFMM:

Multi-seed multi-domain fast-marching method

nM:

Nanomolar

PDB:

Protein data bank

PMSF:

Phenylmethanesulfonyl fluoride or phenylmethylsulfonyl fluoride

RNA:

Ribonucleic acid

rRNA:

Ribosomal ribonucleic acid

SIRT:

Simultaneous iterative reconstruction technique

SKS:

25 mM sucrose, 5 mM KCl

SM:

Semi-defined medium

SNR:

Signal-to-noise ratio

SSU:

Small ribosomal subunit

TLCK:

Tosyllysine chloromethyl ketone hydrochloride

Tris:

Trishydroxymethylaminomethane

tRNA:

Transfer Ribonucleic acid

μm:

Micrometer

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Acknowledgements

We thank G. Cardone for the assistance in the local resolution computation, Dr Harry Kao for managing our computer cluster, and Melissa Thomas for her assistance with the preparation of figures. This work was supported by the Howard Hughes Medical Institute (HHMI) and the National Institutes of Health (NIH) grants R01 GM55440 and R01 GM29169 (to J.F.), L’Agence Nationale de la recherche (ANR) project AMIS ARN ANR-09-BLAN-0160 (E.W. and F.J.), as well as NIH R01-EB004873 and R01-GM074258 (to Q.Z. and C.B.). S.N.B. was supported by Centers for Disease Control (CDC) Emerging Infectious Diseases (EID) Fellowship program.

Author Contributions This chapter was written by A.d.G., Y.H., R. L., and J.F. In the work this chapter is based on, Y.H., A.d.G., C.B, S.M, and J.F. designed the experiments, interpreted the data, and wrote the manuscript published as a letter in Nature [32]. S.N.B. purified the T. brucei ribosomes. Y.H., J. Fu, and R.A.G. carried out the cryo-EM experiments. H.Y.L. performed the three-dimensional variance estimation. Y.H., A.J. C.B., and Q.Z. performed the density-map segmentations. A.d.G., Y.H., R. L., J. Fu, A.J., and H.Y.L. carried out the cryo-EM data processing. Y.H. and F.J. modeled the rRNA. Y.H., C.B., and Q.Z. modeled the ribosomal proteins. J.F. directed the research.

Author Information The electron microscopy map has been deposited in the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute Electron Microscopy Data Bank (EMDB) under accession code EMD-2239. Coordinates of electron-microscopy-based model have been deposited in the RCSB Protein Data Bank under accession numbers 3ZEQ, 3ZEX, 3ZEY, and 3ZF7.

Author information

Authors and Affiliations

  1. Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Amedee des Georges, Yaser Hashem, Robert A. Grassucci & Joachim Frank

  2. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, 10032, USA

    Hstau Y. Liao, Jie Fu & Robert Langlois

  3. New York State Department of Health, Division of Infectious Diseases, Wadsworth Center, Albany, NY, 12201, USA

    Sarah N. Buss & Susan Madison-Antenucci

  4. Architecture et Ractivit de l’ARN, Universit de Strasbourg, Institut de Biologie Molculaire et Cellulaire (CNRS), Strasbourg, 67084, France

    Fabrice Jossinet & Eric Westhof

  5. Department of Biological Sciences, Columbia University, New York, NY, 10027, USA

    Amy Jobe

  6. Department of Computer Science, Institute for Computational Engineering and Sciences, University of Texas, Austin, TX, 78712, USA

    Qin Zhang & Chandrajit Bajaj

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  1. Amedee des Georges
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  14. Joachim Frank
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Correspondence to Amedee des Georges .

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Editors and Affiliations

  1. Department of Computer Science, The Graduate Center City University of New York, New York, New York, USA

    Gabor T. Herman

  2. Department of Biology, Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA

    Joachim Frank

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Georges, A.d. et al. (2014). High-resolution Cryo-EM Structure of the Trypanosoma brucei Ribosome: A Case Study. In: Herman, G., Frank, J. (eds) Computational Methods for Three-Dimensional Microscopy Reconstruction. Applied and Numerical Harmonic Analysis. Birkhäuser, New York, NY. https://doi.org/10.1007/978-1-4614-9521-5_5

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