@article {48644,
	title = {Expression, purification, and contaminant detection for structural studies of Ralstonia metallidurance ClC protein rm1.},
	journal = {PloS One},
	volume = {12},
	year = {2017},
	month = {2017},
	pages = {e0180163},
	abstract = {<p>Single-particle electron cryo-microscopy (cryo-EM) has become a popular method for high-resolution study of the structural and functional properties of proteins. However, sufficient expression and purification of membrane proteins holds many challenges. We describe methods to overcome these obstacles using ClC-rm1, a prokaryotic chloride channel (ClC) family protein from Ralstonia metallidurans, overexpressed in Escherichia coli (E. coli) BL21(DE3) strain. Mass spectrometry and electron microscopy analyses of purified samples revealed multiple contaminants that can obfuscate results of subsequent high-resolution structural analysis. Here we describe the systematic optimization of sample preparation procedures, including expression systems, solubilization techniques, purification protocols, and contamination detection. We found that expressing ClC-rm1 in E. coli BL21(DE3) and using n-dodecyl-β-D-maltopyranoside as a detergent for solubilization and purification steps resulted in the highest quality samples of those we tested. However, although protein yield, sample stability, and the resolution of structural detail were improved following these changes, we still detected contaminants including Acriflavine resistant protein AcrB. AcrB was particularly difficult to remove as it co-purified with ClC-rm1 due to four intrinsic histidine residues at its C-terminus that bind to affinity resins. We were able to obtain properly folded pure ClC-rm1 by adding eGFP to the C-terminus and overexpressing the protein in the ΔacrB variant of the JW0451-2 E. coli strain.</p>
},
	issn = {1932-6203},
	doi = {10.1371/journal.pone.0180163},
	author = {Abeyrathne, Priyanka D and Grigorieff, Nikolaus}
}
@article {47465,
	title = {Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome.},
	journal = {eLife},
	volume = {5},
	year = {2016},
	month = {2016 May 9},
	abstract = {<p>Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2{\textbullet}GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation.</p>
},
	issn = {2050-084X},
	doi = {10.7554/eLife.14874},
	author = {Abeyrathne, Priyanka D and Koh, Cha San and Grant, Timothy and Grigorieff, Nikolaus and Korostelev, Andrei A}
}
