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PDBsum entry 6o6c
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Transferase/DNA/RNA
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PDB id
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6o6c
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Contents |
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1446 a.a.
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1165 a.a.
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270 a.a.
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215 a.a.
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81 a.a.
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146 a.a.
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115 a.a.
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65 a.a.
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111 a.a.
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46 a.a.
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References listed in PDB file
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Key reference
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Title
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3.1 å structure of yeast RNA polymerase ii elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids.
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Authors
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I.Lahiri,
J.Xu,
B.G.Han,
J.Oh,
D.Wang,
F.Dimaio,
A.E.Leschziner.
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Ref.
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J Struct Biol, 2019,
207,
270-278.
[DOI no: ]
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PubMed id
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Abstract
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Despite significant advances in all aspects of single particle cryo-electron
microscopy (cryo-EM), specimen preparation still remains a challenge. During
sample preparation, macromolecules interact with the air-water interface, which
often leads to detrimental effects such as denaturation or adoption of preferred
orientations, ultimately hindering structure determination. Randomly
biotinylating the protein of interest (for example, at its primary amines) and
then tethering it to a cryo-EM grid coated with two-dimensional crystals of
streptavidin (acting as an affinity surface) can prevent the protein from
interacting with the air-water interface. Recently, this approach was
successfully used to solve a high-resolution structure of a test sample, a
bacterial ribosome. However, whether this method can be used for samples where
interaction with the air-water interface has been shown to be problematic
remains to be determined. Here we report a 3.1 Å structure of an RNA
polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer
lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to
solve this structure using conventional sample preparation methods resulted in a
poor quality cryo-EM map due to Pol II EC(CPD)'s adopting a strong preferred
orientation. Imaging the same sample on streptavidin grids improved the angular
distribution of its view, resulting in a high-resolution structure. This work
shows that streptavidin affinity grids can be used to address known challenges
posed by the interaction with the air-water interface.
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