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Rational design of true monomeric and bright photoactivatable fluorescent proteins


Monomeric (m)Eos2 is an engineered photoactivatable fluorescent protein widely used for super-resolution microscopy. We show that mEos2 forms oligomers at high concentrations and forms aggregates when labeling membrane proteins, limiting its application as a fusion partner. We solved the crystal structure of tetrameric mEos2 and rationally designed improved versions, mEos3.1 and mEos3.2, that are truly monomeric, are brighter, mature faster and exhibit higher photon budget and label density.

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Figure 1: mEos3.1 and 2 are true monomeric fluorescent proteins.
Figure 2: Comparing the label density and Nyquist resolution of EosFP variants and mClavGR2.

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We thank L.L. Looger (Janelia Farm Research Campus) and Addgene for providing mEos2 cDNA, and X. Yu for providing technical support of analytical ultracentrifugation. This work was supported by grants from the Major State Basic Research Program of the People's Republic of China (2010CB833701 and 2010CB912303), the National Science Foundation of China (31130065, 31170818, 90913022 and 31127901), projects from Chinese Academy of Sciences (YZ200838, KSCX1-1W-J-3 and KSCX2-EW-Q-11), and the talent introduction program to Universities (B08029).

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Authors and Affiliations



M.Z., H.C., Y.Z., J.Y., W.J., J.C., B.L. and J.L. performed the research; L.W. and Y.L. assisted with data collection and solved the mEos2 structure; J.Z. assisted with spectrum measurement; M.Z., H.C., Y.Z., P.X. and T.X. analyzed data; T.X. and P.X. designed the research and wrote the paper.

Corresponding authors

Correspondence to Pingyong Xu or Tao Xu.

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The authors declare no competing financial interests.

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Supplementary Figures 1–17 and Supplementary Tables 1–7 (PDF 2305 kb)

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Zhang, M., Chang, H., Zhang, Y. et al. Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat Methods 9, 727–729 (2012).

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