Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
09 12 2021
09 12 2021
Historique:
received:
19
02
2021
accepted:
12
11
2021
entrez:
10
12
2021
pubmed:
11
12
2021
medline:
20
1
2022
Statut:
epublish
Résumé
Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.
Identifiants
pubmed: 34887415
doi: 10.1038/s41467-021-27400-7
pii: 10.1038/s41467-021-27400-7
pmc: PMC8660796
doi:
Substances chimiques
Viral Proteins
0
virus protein 2A
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
7166Subventions
Organisme : Wellcome Trust (Wellcome)
ID : 202797/Z/16/Z
Organisme : Wellcome Trust (Wellcome)
ID : 221818/Z/20/Z
Organisme : Wellcome Trust
ID : WT096570
Pays : United Kingdom
Organisme : Wellcome Trust (Wellcome)
ID : 106207/Z/14/Z
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/D009499/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_U105184332
Pays : United Kingdom
Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : Wellcome Trust (Wellcome)
ID : 098406/Z/12/B
Informations de copyright
© 2021. The Author(s).
Références
Firth, A. E. & Brierley, I. Non-canonical translation in RNA viruses. J. Gen. Virol. 93, 1385–1409 (2012).
pubmed: 22535777
pmcid: 3542737
doi: 10.1099/vir.0.042499-0
Atkins, J. F., Loughran, G., Bhatt, P. R., Firth, A. E. & Baranov, P. V. Ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use. Nucleic Acids Res. 44, 7007–7078 (2016).
pubmed: 27436286
pmcid: 5009743
Korniy, N., Samatova, E., Anokhina, M. M., Peske, F. & Rodnina, M. V. Mechanisms and biomedical implications of -1 programmed ribosome frameshifting on viral and bacterial mRNAs. FEBS Lett. 593, 1468–1482 (2019).
pubmed: 31222875
pmcid: 6771820
doi: 10.1002/1873-3468.13478
Chen, J. et al. Dynamic pathways of -1 translational frameshifting. Nature 512, 328–332 (2014).
pubmed: 24919156
pmcid: 4472451
doi: 10.1038/nature13428
Caliskan, N., Katunin, V. I., Belardinelli, R., Peske, F. & Rodnina, M. V. Programmed -1 frameshifting by kinetic partitioning during impeded translocation. Cell 157, 1619–1631 (2014).
pubmed: 24949973
pmcid: 7112342
doi: 10.1016/j.cell.2014.04.041
Choi, J., O’Loughlin, S., Atkins, J. F. & Puglisi, J. D. The energy landscape of -1 ribosomal frameshifting. Sci. Adv. 6, eaax6969 (2020).
pubmed: 31911945
pmcid: 6938710
doi: 10.1126/sciadv.aax6969
Namy, O., Moran, S. J., Stuart, D. I., Gilbert, R. J. & Brierley, I. A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting. Nature 441, 244–247 (2006).
pubmed: 16688178
pmcid: 7094908
doi: 10.1038/nature04735
Giedroc, D. P. & Cornish, P. V. Frameshifting RNA pseudoknots: structure and mechanism. Virus Res. 139, 193–208 (2009).
pubmed: 18621088
doi: 10.1016/j.virusres.2008.06.008
Chen, G., Chang, K. Y., Chou, M. Y., Bustamante, C. & Tinoco, I. Jr. Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of -1 ribosomal frameshifting. Proc. Natl Acad. Sci. USA 106, 12706–12711 (2009).
pubmed: 19628688
pmcid: 2722267
doi: 10.1073/pnas.0905046106
Halma, M. T. J., Ritchie, D. B., Cappellano, T. R., Neupane, K. & Woodside, M. T. Complex dynamics under tension in a high-efficiency frameshift stimulatory structure. Proc. Natl Acad. Sci. USA 116, 19500–19505 (2019).
pubmed: 31409714
pmcid: 6765238
doi: 10.1073/pnas.1905258116
Loughran, G., Firth, A. E. & Atkins, J. F. Ribosomal frameshifting into an overlapping gene in the 2B-encoding region of the cardiovirus genome. Proc. Natl Acad. Sci. USA 108, E1111–E1119 (2011).
pubmed: 22025686
pmcid: 3219106
doi: 10.1073/pnas.1102932108
Napthine, S. et al. Protein-directed ribosomal frameshifting temporally regulates gene expression. Nat. Commun. 8, 15582 (2017).
pubmed: 28593994
pmcid: 5472766
doi: 10.1038/ncomms15582
Jackson, R. J. A detailed kinetic analysis of the in vitro synthesis and processing of encephalomyocarditis virus products. Virology 149, 114–127 (1986).
pubmed: 3004023
doi: 10.1016/0042-6822(86)90092-9
Hahn, H. & Palmenberg, A. C. Deletion mapping of the encephalomyocarditis virus primary cleavage site. J. Virol. 75, 7215–7218 (2001).
pubmed: 11435606
pmcid: 114454
doi: 10.1128/JVI.75.15.7215-7218.2001
Yang, X. et al. Structures and corresponding functions of five types of picornaviral 2A proteins. Front. Microbiol. 8, 1373 (2017).
pubmed: 28785248
pmcid: 5519566
doi: 10.3389/fmicb.2017.01373
Groppo, R. & Palmenberg, A. C. Cardiovirus 2A protein associates with 40S but not 80S ribosome subunits during infection. J. Virol. 81, 13067–13074 (2007).
pubmed: 17728235
pmcid: 2169094
doi: 10.1128/JVI.00185-07
Carocci, M. et al. Encephalomyocarditis virus 2A protein is required for viral pathogenesis and inhibition of apoptosis. J. Virol. 85, 10741–10754 (2011).
pubmed: 21849462
pmcid: 3187497
doi: 10.1128/JVI.00394-11
Merrick, W. C. eIF4F: a retrospective. J. Biol. Chem. 290, 24091–24099 (2015).
pubmed: 26324716
pmcid: 4591800
doi: 10.1074/jbc.R115.675280
Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D. Biol. Crystallogr. 60, 2256–2268 (2004).
pubmed: 15572779
doi: 10.1107/S0907444904026460
Holm, L. & Laakso, L. M. Dali server update. Nucleic Acids Res. 44, W351–W355 (2016).
pubmed: 27131377
pmcid: 4987910
doi: 10.1093/nar/gkw357
Redfern, O. C., Harrison, A., Dallman, T., Pearl, F. M. & Orengo, C. A. CATHEDRAL: a fast and effective algorithm to predict folds and domain boundaries from multidomain protein structures. PLoS Comput. Biol. 3, e232 (2007).
pubmed: 18052539
pmcid: 2098860
doi: 10.1371/journal.pcbi.0030232
Svitkin, Y. V., Hahn, H., Gingras, A. C., Palmenberg, A. C. & Sonenberg, N. Rapamycin and wortmannin enhance replication of a defective encephalomyocarditis virus. J. Virol. 72, 5811–5819 (1998).
pubmed: 9621041
pmcid: 110383
doi: 10.1128/JVI.72.7.5811-5819.1998
Groppo, R., Brown, B. A. & Palmenberg, A. C. Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site. Virology 410, 257–267 (2011).
pubmed: 21145089
doi: 10.1016/j.virol.2010.11.002
Petty, R. V., Basta, H. A., Bacot-Davis, V. R., Brown, B. A. & Palmenberg, A. C. Binding interactions between the encephalomyocarditis virus leader and protein 2A. J. Virol. 88, 13503–13509 (2014).
pubmed: 25210192
pmcid: 4249092
doi: 10.1128/JVI.02148-14
Siddiqui, N. et al. Structural insights into the allosteric effects of 4EBP1 on the eukaryotic translation initiation factor eIF4E. J. Mol. Biol. 415, 781–792 (2012).
pubmed: 22178476
doi: 10.1016/j.jmb.2011.12.002
Magnus, M., Boniecki, M. J., Dawson, W. & Bujnicki, J. M. SimRNAweb: a web server for RNA 3D structure modeling with optional restraints. Nucleic Acids Res. 44, W315–W319 (2016).
pubmed: 27095203
pmcid: 4987879
doi: 10.1093/nar/gkw279
Ren, J., Rastegari, B., Condon, A. & Hoos, H. H. HotKnots: heuristic prediction of RNA secondary structures including pseudoknots. RNA 11, 1494–1504 (2005).
pubmed: 16199760
pmcid: 1370833
doi: 10.1261/rna.7284905
Chen, G., Wen, J. D. & Tinoco, I. Jr. Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA. RNA 13, 2175–2188 (2007).
pubmed: 17959928
pmcid: 2080604
doi: 10.1261/rna.676707
Li, P. T., Collin, D., Smith, S. B., Bustamante, C. & Tinoco, I. Jr. Probing the mechanical folding kinetics of TAR RNA by hopping, force-jump, and force-ramp methods. Biophys. J. 90, 250–260 (2006).
pubmed: 16214869
doi: 10.1529/biophysj.105.068049
Heller, I., Hoekstra, T. P., King, G. A., Peterman, E. J. & Wuite, G. J. Optical tweezers analysis of DNA-protein complexes. Chem. Rev. 114, 3087–3119 (2014).
pubmed: 24443844
doi: 10.1021/cr4003006
Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).
pubmed: 12824337
pmcid: 169194
doi: 10.1093/nar/gkg595
Ritchie, D. B., Foster, D. A. & Woodside, M. T. Programmed -1 frameshifting efficiency correlates with RNA pseudoknot conformational plasticity, not resistance to mechanical unfolding. Proc. Natl Acad. Sci. USA 109, 16167–16172 (2012).
pubmed: 22988073
pmcid: 3479558
doi: 10.1073/pnas.1204114109
Mangeol, P. et al. Probing ribosomal protein-RNA interactions with an external force. Proc. Natl Acad. Sci. USA 108, 18272–18276 (2011).
pubmed: 22025688
pmcid: 3215066
doi: 10.1073/pnas.1107121108
Liphardt, J., Dumont, S., Smith, S. B., Tinoco, I. Jr. & Bustamante, C. Equilibrium information from nonequilibrium measurements in an experimental test of Jarzynski’s equality. Science 296, 1832–1835 (2002).
pubmed: 12052949
doi: 10.1126/science.1071152
Leger, M., Sidani, S. & Brakier-Gingras, L. A reassessment of the response of the bacterial ribosome to the frameshift stimulatory signal of the human immunodeficiency virus type 1. RNA 10, 1225–1235 (2004).
pubmed: 15247429
pmcid: 1370612
doi: 10.1261/rna.7670704
Horsfield, J. A., Wilson, D. N., Mannering, S. A., Adamski, F. M. & Tate, W. P. Prokaryotic ribosomes recode the HIV-1 gag-pol-1 frameshift sequence by an E/P site post-translocation simultaneous slippage mechanism. Nucleic Acids Res. 23, 1487–1494 (1995).
pubmed: 7784201
pmcid: 306887
doi: 10.1093/nar/23.9.1487
James, N. R., Brown, A., Gordiyenko, Y. & Ramakrishnan, V. Translational termination without a stop codon. Science 354, 1437–1440 (2016).
pubmed: 27934701
pmcid: 5351859
doi: 10.1126/science.aai9127
Brilot, A. F., Korostelev, A. A., Ermolenko, D. N. & Grigorieff, N. Structure of the ribosome with elongation factor G trapped in the pretranslocation state. Proc. Natl Acad. Sci. USA 110, 20994–20999 (2013).
pubmed: 24324137
pmcid: 3876248
doi: 10.1073/pnas.1311423110
Lin, J., Gagnon, M. G., Bulkley, D. & Steitz, T. A. Conformational changes of elongation factor G on the ribosome during tRNA translocation. Cell 160, 219–227 (2015).
pubmed: 25594181
pmcid: 4297320
doi: 10.1016/j.cell.2014.11.049
Bulkley, D. et al. The antibiotics dityromycin and GE82832 bind protein S12 and block EF-G-catalyzed translocation. Cell Rep. 6, 357–365 (2014).
pubmed: 24412368
pmcid: 5331365
doi: 10.1016/j.celrep.2013.12.024
Fislage, M. et al. Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-TuH84A. Nucleic Acids Res. 46, 5861–5874 (2018).
pubmed: 29733411
pmcid: 6009598
doi: 10.1093/nar/gky346
Loveland, A. B., Demo, G. & Korostelev, A. A. Cryo-EM of elongating ribosome with EF-Tu*GTP elucidates tRNA proofreading. Nature 584, 640–645 (2020).
Hussain, T., Llacer, J. L., Wimberly, B. T., Kieft, J. S. & Ramakrishnan, V. Large-scale movements of IF3 and tRNA during bacterial translation initiation. Cell 167, 133–144.e13 (2016).
pubmed: 27662086
pmcid: 5037330
doi: 10.1016/j.cell.2016.08.074
Napthine, S., Bell, S., Hill, C. H., Brierley, I. & Firth, A. E. Characterization of the stimulators of protein-directed ribosomal frameshifting in Theiler’s murine encephalomyelitis virus. Nucleic Acids Res. 47, 8207–8223 (2019).
pubmed: 31180502
pmcid: 6735917
doi: 10.1093/nar/gkz503
Wen, J. D. et al. Following translation by single ribosomes one codon at a time. Nature 452, 598–603 (2008).
pubmed: 18327250
pmcid: 2556548
doi: 10.1038/nature06716
Kim, H. K. et al. A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation. Proc. Natl Acad. Sci. USA 111, 5538–5543 (2014).
pubmed: 24706807
pmcid: 3992627
doi: 10.1073/pnas.1403457111
Qin, P., Yu, D., Zuo, X. & Cornish, P. V. Structured mRNA induces the ribosome into a hyper-rotated state. EMBO Rep. 15, 185–190 (2014).
pubmed: 24401932
pmcid: 3989864
doi: 10.1002/embr.201337762
Bao, C. et al. mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding. eLife 9, e55799 (2020).
Yang, L. et al. Single-molecule mechanical folding and unfolding of RNA hairpins: effects of single A-U to A.C pair substitutions and single proton binding and implications for mRNA structure-induced -1 ribosomal frameshifting. J. Am. Chem. Soc. 140, 8172–8184 (2018).
pubmed: 29884019
doi: 10.1021/jacs.8b02970
Zhong, Z. et al. Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for -1 ribosomal frameshifting stimulation. Sci. Rep. 6, 39549 (2016).
pubmed: 28000744
pmcid: 5175198
doi: 10.1038/srep39549
Bock, L. V. et al. Thermodynamic control of -1 programmed ribosomal frameshifting. Nat. Commun. 10, 4598 (2019).
pubmed: 31601802
pmcid: 6787027
doi: 10.1038/s41467-019-12648-x
Peng, B. Z. et al. Active role of elongation factor G in maintaining the mRNA reading frame during translation. Sci. Adv. 5, eaax8030 (2019).
pubmed: 31903418
pmcid: 6924986
doi: 10.1126/sciadv.aax8030
Neidel, S. et al. Vaccinia virus protein A49 is an unexpected member of the B-cell Lymphoma (Bcl)-2 protein family. J. Biol. Chem. 290, 5991–6002 (2015).
pubmed: 25605733
pmcid: 4358236
doi: 10.1074/jbc.M114.624650
Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Cryst. 43, 186–190 (2009).
doi: 10.1107/S0021889809045701
Kabsch, W. XDS. Acta Crystallogr. D. Biol. Crystallogr. 66, 125–132 (2010).
pubmed: 20124692
pmcid: 2815665
doi: 10.1107/S0907444909047337
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D. Biol. Crystallogr. 69, 1204–1214 (2013).
pubmed: 23793146
pmcid: 3689523
doi: 10.1107/S0907444913000061
Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).
pubmed: 22628654
pmcid: 3457925
doi: 10.1126/science.1218231
Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007).
pubmed: 17172768
Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).
pubmed: 18156677
doi: 10.1107/S0108767307043930
Abrahams, J. P. & Leslie, A. G. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D. Biol. Crystallogr. 52, 30–42 (1996).
pubmed: 15299723
doi: 10.1107/S0907444995008754
Perrakis, A., Harkiolaki, M., Wilson, K. S. & Lamzin, V. S. ARP/wARP and molecular replacement. Acta Crystallogr. D. Biol. Crystallogr. 57, 1445–1450 (2001).
pubmed: 11567158
doi: 10.1107/S0907444901014007
McCoy, A. J. et al. Phaser crystallographic software. J. Appl Crystallogr 40, 658–674 (2007).
pubmed: 19461840
pmcid: 2483472
doi: 10.1107/S0021889807021206
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).
pubmed: 20383002
pmcid: 2852313
doi: 10.1107/S0907444910007493
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).
pubmed: 20124702
pmcid: 2815670
doi: 10.1107/S0907444909052925
Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta. Crystallogr. D. Struct. Biol. 74, 519–530 (2018).
pubmed: 29872003
pmcid: 6096486
doi: 10.1107/S2059798318002425
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D. Biol. Crystallogr. 66, 12–21 (2010).
pubmed: 20057044
doi: 10.1107/S0907444909042073
Dolinsky, T. J., Nielsen, J. E., McCammon, J. A. & Baker, N. A. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 32, W665–W667 (2004).
pubmed: 15215472
pmcid: 441519
doi: 10.1093/nar/gkh381
Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001).
pubmed: 11517324
pmcid: 56910
doi: 10.1073/pnas.181342398
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
pubmed: 17681537
doi: 10.1016/j.jmb.2007.05.022
Stephenson, W., Wan, G., Tenenbaum, S. A. & Li, P. T. Nanomanipulation of single RNA molecules by optical tweezers. J. Vis. Exp. 51542 (2014).
Mukhortava, A. et al. Structural heterogeneity of attC integron recombination sites revealed by optical tweezers. Nucleic Acids Res. 47, 1861–1870 (2019).
pubmed: 30566629
doi: 10.1093/nar/gky1258
Wang, M. D., Yin, H., Landick, R., Gelles, J. & Block, S. M. Stretching DNA with optical tweezers. Biophys. J. 72, 1335–1346 (1997).
pubmed: 9138579
pmcid: 1184516
doi: 10.1016/S0006-3495(97)78780-0
Odijk, T. Stiff chains and filaments under tension. Macromolecules 28, 7016–7018 (1995).
doi: 10.1021/ma00124a044
Zhang, C. et al. The mechanical properties of RNA-DNA hybrid duplex stretched by magnetic tweezers. Biophys. J. 116, 196–204 (2019).
pubmed: 30635125
doi: 10.1016/j.bpj.2018.12.005
Smith, S. B., Cui, Y. & Bustamante, C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271, 795–799 (1996).
pubmed: 8628994
doi: 10.1126/science.271.5250.795
McCauley, M. J., Rouzina, I., Li, J., Nunez, M. E. & Williams, M. C. Significant differences in RNA structure destabilization by HIV-1 GagDp6 and NCp7 proteins. Viruses 12, 484 (2020).
Gore, J., Ritort, F. & Bustamante, C. Bias and error in estimates of equilibrium free-energy differences from nonequilibrium measurements. Proc. Natl Acad. Sci. USA 100, 12564–12569 (2003).
pubmed: 14528008
pmcid: 240657
doi: 10.1073/pnas.1635159100
Bellaousov, S., Reuter, J. S., Seetin, M. G. & Mathews, D. H. RNAstructure: Web servers for RNA secondary structure prediction and analysis. Nucleic Acids Res. 41, W471–W474 (2013).
pubmed: 23620284
pmcid: 3692136
doi: 10.1093/nar/gkt290
Pisarev, A. V., Unbehaun, A., Hellen, C. U. & Pestova, T. V. Assembly and analysis of eukaryotic translation initiation complexes. Methods Enzymol. 430, 147–177 (2007).
pubmed: 17913638
doi: 10.1016/S0076-6879(07)30007-4
Fixsen, S. M. & Howard, M. T. Processive selenocysteine incorporation during synthesis of eukaryotic selenoproteins. J. Mol. Biol. 399, 385–396 (2010).
pubmed: 20417644
pmcid: 2916059
doi: 10.1016/j.jmb.2010.04.033
Powell, M. L., Brown, T. D. & Brierley, I. Translational termination-re-initiation in viral systems. Biochem. Soc. Trans. 36, 717–722 (2008).
pubmed: 18631147
doi: 10.1042/BST0360717
Milon, P. et al. Transient kinetics, fluorescence, and FRET in studies of initiation of translation in bacteria. Methods Enzymol. 430, 1–30 (2007).
pubmed: 17913632
doi: 10.1016/S0076-6879(07)30001-3
Rodnina, M. V., Semenkov, Y. P. & Wintermeyer, W. Purification of fMet-tRNA(fMet) by fast protein liquid chromatography. Anal. Biochem. 219, 380–381 (1994).
pubmed: 8080098
doi: 10.1006/abio.1994.1282
Kothe, U., Paleskava, A., Konevega, A. L. & Rodnina, M. V. Single-step purification of specific tRNAs by hydrophobic tagging. Anal. Biochem. 356, 148–150 (2006).
pubmed: 16750812
doi: 10.1016/j.ab.2006.04.038
Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89 (1990).
pubmed: 2199796
doi: 10.1016/0076-6879(90)85008-C
Passmore, L. A. & Russo, C. J. Specimen preparation for high-resolution cryo-EM. Methods Enzymol. 579, 51–86 (2016).
pubmed: 27572723
pmcid: 5140023
doi: 10.1016/bs.mie.2016.04.011
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
pubmed: 28250466
pmcid: 5494038
doi: 10.1038/nmeth.4193
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
pubmed: 26278980
pmcid: 6760662
doi: 10.1016/j.jsb.2015.08.008
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
pubmed: 15264254
doi: 10.1002/jcc.20084