@article{618cbd9332ee400092f4da3034090e68,
title = "Assembly and Cryo-EM structure determination of yeast mitochondrial RNA polymerase initiation complex intermediates",
abstract = "In yeast mitochondria, transcription initiation requires assembly of mitochondrial RNA polymerase and transcription initiation factor MTF1 at the DNA promoter initiation site. This protocol describes the purification of partially melted and fully melted initiation complex states. Both states co-exist in equilibrium in the same sample as seen by cryoelectron microscopy (cryo-EM) and allow elucidation of MTF1's structural roles in controlling the transition into elongation. We further outline how analysis of the complex by light scattering, thermal shift assay, and ultrafiltration assay exhibits reproducible results.",
keywords = "Cryo-EM, Protein expression and purification, Structural Biology",
author = "Martinez, {Sergio E.} and Anupam Singh and {De Wijngaert}, Brent and Shemaila Sultana and Chhaya Dharia and Hans Vanbuel and Jiayu Shen and Daniel Vasilchuk and Patel, {Smita S.} and Kalyan Das",
note = "Funding Information: We thank the Electron Cryogenic Microscopy (Brussels), Thermo Fisher Scientific (Eindhoven), and ESRF Grenoble for microscope access as well as Marcus Fislage, Adrian Koh, and Abhimanyu Singh for helpful discussion. This research was supported by NIH grant R35 GM118086 to S.S.P. and KU Leuven start-up and Rega Virology and Chemotherapy internal grants to K.D. Funding Information: We thank the Electron Cryogenic Microscopy (Brussels), Thermo Fisher Scientific (Eindhoven), and ESRF Grenoble for microscope access as well as Marcus Fislage, Adrian Koh, and Abhimanyu Singh for helpful discussion. This research was supported by NIH grant R35 GM118086 to S.S.P. and KU Leuven start-up and Rega Virology and Chemotherapy internal grants to K.D. Investigation and validation, B.D.W. S.S. A.S. H.V. J.S. D.V. S.E.M. and K.D.; conceptualization, B.D.W. S.S. S.S.P. and K.D.; resources, C.D. and E.K.; data curation, B.D.W. E.K. S.S.P. and K.D.; writing and editing S.E.M. S.S.P. and K.D. supervision, visualization, funding acquisition, and project administration, S.S.P. and K.D. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2021 The Author(s)",
year = "2021",
month = jun,
day = "18",
doi = "10.1016/j.xpro.2021.100431",
language = "English (US)",
volume = "2",
journal = "STAR Protocols",
issn = "2666-1667",
publisher = "Cell Press",
number = "2",
}