Preparation of RNA Polymerase Complexes for Their Analysis by Single-Particle Cryo-Electron Microscopy.
Grid preparation
Negative staining
Plunge freezing
Preparation of transcription complexes
RNA polymerase I
Single-particle cryo-electron microscopy
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
7
7
2022
pubmed:
8
7
2022
medline:
12
7
2022
Statut:
ppublish
Résumé
Recent technological progress revealed new prospects of high-resolution structure determination of macromolecular complexes using cryo-electron microscopy (cryo-EM) . In the field of RNA polymerase (Pol) I research, a number of cryo-EM studies contributed to understanding the highly specialized mechanisms underlying the transcription of ribosomal RNA genes . Despite a broad applicability of the cryo-EM method itself, preparation of samples for high-resolution data collection can be challenging. Here, we describe strategies for the purification and stabilization of Pol I complexes, exemplarily considering advantages and disadvantages of the methodology. We further provide an easy-to-implement protocol for the coating of EM-grids with self-made carbon support films. In sum, we present an efficient workflow for cryo-grid preparation and optimization, including early stage cryo-EM screening that can be adapted to a wide range of soluble samples for high-resolution structure determination .
Identifiants
pubmed: 35796984
doi: 10.1007/978-1-0716-2501-9_6
pmc: PMC9761500
doi:
Substances chimiques
Macromolecular Substances
0
DNA-Directed RNA Polymerases
EC 2.7.7.6
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
81-96Informations de copyright
© 2022. The Author(s).
Références
J Biol Chem. 2019 Mar 29;294(13):5181-5197
pubmed: 30804214
Nature. 2016 Dec 22;540(7634):607-610
pubmed: 27842382
Nature. 2013 Mar 28;495(7442):481-6
pubmed: 23446344
Nat Methods. 2017 Mar;14(3):290-296
pubmed: 28165473
Mol Cell. 2016 Dec 15;64(6):1135-1143
pubmed: 27867008
J Struct Biol. 2018 Nov;204(2):270-275
pubmed: 30055234
Nat Commun. 2021 Feb 3;12(1):758
pubmed: 33536435
Nat Commun. 2016 Jul 15;7:12126
pubmed: 27418187
J Struct Biol. 2005 Oct;152(1):36-51
pubmed: 16182563
Annu Rev Biophys. 2019 May 6;48:45-61
pubmed: 30786229
Nat Methods. 2016 Jan;13(1):24-7
pubmed: 27110629
Cell. 2017 Mar 23;169(1):120-131.e22
pubmed: 28340337
Commun Biol. 2019 Jun 19;2:218
pubmed: 31240256
Elife. 2017 Mar 06;6:
pubmed: 28262097
Nat Methods. 2008 Jan;5(1):53-5
pubmed: 18157137
Curr Opin Struct Biol. 2018 Oct;52:8-15
pubmed: 30015202
Methods Enzymol. 2010;481:109-26
pubmed: 20887855
Rev Sci Instrum. 2016 Nov;87(11):114302
pubmed: 27910462
Nature. 2013 Oct 31;502(7473):644-9
pubmed: 24153184
Elife. 2019 Apr 01;8:
pubmed: 30932812
Q Rev Biophys. 1988 May;21(2):129-228
pubmed: 3043536
J Struct Biol. 2010 Apr;170(1):152-6
pubmed: 20035878
Nat Methods. 2019 Jun;16(6):471-477
pubmed: 31086343
Nature. 2015 Sep 10;525(7568):172-4
pubmed: 26354465
Methods Mol Biol. 2016;1455:85-97
pubmed: 27576712
Methods Enzymol. 2016;579:51-86
pubmed: 27572723
Methods. 2016 May 1;100:3-15
pubmed: 26931652
Proc Natl Acad Sci U S A. 2018 Sep 4;115(36):8972-8977
pubmed: 30127008
J Struct Biol. 2011 Apr;174(1):234-8
pubmed: 20937392
Methods Mol Biol. 2011;718:3-22
pubmed: 21370039
Nature. 2013 Oct 31;502(7473):650-5
pubmed: 24153182
Nat Commun. 2020 Mar 5;11(1):1206
pubmed: 32139698
Nature. 2016 Sep 14;537(7620):339-46
pubmed: 27629640
Elife. 2018 Nov 09;7:
pubmed: 30412051
J Struct Biol. 2018 Oct;204(1):80-84
pubmed: 30017701
Nat Commun. 2016 Jul 15;7:12129
pubmed: 27418309
Nat Methods. 2019 Nov;16(11):1146-1152
pubmed: 31591575
Nat Methods. 2014 Jun;11(6):649-52
pubmed: 24747813
Nat Protoc. 2007;2(12):3239-46
pubmed: 18079724
Methods Mol Biol. 2016;1455:99-108
pubmed: 27576713
Science. 2014 Mar 28;343(6178):1443-4
pubmed: 24675944
Nat Protoc. 2008;3(2):330-9
pubmed: 18274535
Q Rev Biophys. 2016 Jan;49:e13
pubmed: 27658821
Nat Protoc. 2017 Feb;12(2):209-212
pubmed: 28055037
Methods Enzymol. 2016;579:87-102
pubmed: 27572724
Microscopy (Oxf). 2016 Feb;65(1):23-34
pubmed: 26671943
Elife. 2017 Jun 17;6:
pubmed: 28623663