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.
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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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