mEos4a

a.k.a. mEos3dG16

mEos4a is a photoconvertible red fluorescent protein published in 2015, derived from Lobophyllia hemprichii. It has moderate acid sensitivity.

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Oligomerization	Organism	Molecular Weight	Cofactor
Monomer	Lobophyllia hemprichii	26.0 kDa	-

FPbase ID: EC56T

Attributes

State	Ex λ	Em λ	EC (M^-1 cm^-1)	QY	Brightness	pKa	Maturation (min)	Lifetime (ns)
Green	505	515	83,530	0.86	71.84	5.25
Red	571	580	61,000	0.71	43.31	5.7

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Transitions

From	To	Switch λ
Green	Red	405

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mEos4a OSER Measurements

% Normal Cells	OSER/NE ratio	Cell Type	Reference
54.0 ± 6.8 (10000 cells)	-	HeLa	Paez-Segala et al. (2015)

Photostability

No photostability measurements available ... add one!

mEos4a Sequence

BLAST

mEos4a was derived from mEos2 with the following mutations: M1_S2insV/K9R/F34Y/S39T/A69V/I102Y/C195AM1_S2insV/K9R/F34Y/S39T/A69V/I102Y/C195A

Note: Figure Sup. 6A of the original paper (Paez-Segala et al. 2015) erroneously shows mEos4a as lacking Met-14. The sequence below includes M14 and should be considered the correct sequence (Paez-Segala, personal communication)

MVSAIKPDMRIKLRMEGNVNGHHFVIDGDGTGKPYEGKQTMDLEVKEGGPLPFAFDILTTAFHYGNRVFVKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICYARNDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIHMALLLEGNAHYRCDFRTTYKAKEKGVKLPGYHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLPDNARR

Excerpts

We mutated surface residues on mEos2 to remove nucleophilic groups, which are involved in cross-linking with aldehydes and OsO4. This resulted in the selection of two mutants, mEos4a and mEos4b, each with substantially improved resistance to OsO4 fixation and fluorescence properties unchanged from those of the starting scaffold mEos2. These proteins facilitated the development of two protocols: (i) a ‘consecutive-section’ approach in which adjacent ultrathin sections cut from resin are separately split between PALM imaging and EM fixation and imaging; and (ii) a ‘same-section’ approach, in which a single resin-cut section is subjected to both PALM and TEM and/or SEM. In both cases, plastic resin embedding markedly decreases tissue distortion from dehydration and secondary fixation, and additionally improves performance of the specimen under the electron beam. We also found that this protocol is appropriate for use with HPF-FS (e.g., Fig. 1d) or without it (e.g., Fig. 1e). We found ultrastructure preservation to be comparable between the two.

Kopek et al. (2017)

Osmium-resistant probes for fluorescence and electron microscopy—the fluorescent EosFP derivative mEos4 has recently been described to withstand osmium treatment during EM processing. The mEos4 molecule is therefore still fluorescent in a well-stained resin-embedded sample. In addition, it is compatible with super resolution photoactivated localization microscopy.

Ando et al. (2018)