PDBsum entry 3ekj

Fluorescent protein

PDB id

3ekj

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Contents

			Protein chain

					302 a.a.

			Waters ×5

PDB id:

3ekj

Links

Name:

Fluorescent protein

Title:

Calcium-free gcamp2 (calcium binding deficient mutant)

Structure:

Myosin light chain kinase, green fluorescent protein, calmodulin chimera. Chain: a. Engineered: yes. Mutation: yes

Source:

Artificial gene, aequorea victoria, rattus norvegicus. Jellyfish, rat. Organism_taxid: 32630, 6100, 10116. Gene: gfp, calm1, calm, cam, cam1, cami. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

2.80Å

R-factor:

0.213

R-free:

0.280

Authors:

J.Akerboom,J.D.Velez Rivera,L.L.Looger,E.R.Schreiter

Key ref:

J.Akerboom et al. (2009). Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. J Biol Chem, 284, 6455-6464. PubMed id: 19098007 DOI: 10.1074/jbc.M807657200

Date:

19-Sep-08

Release date:

16-Dec-08

PROCHECK

	Headers

	References

Protein chain

P0DP29 (CALM1_RAT) - Calmodulin-1 from Rattus norvegicus

Seq: Struc:					149 a.a. 302 a.a.*

Protein chain

P11799 (MYLK_CHICK) - Myosin light chain kinase, smooth muscle from Gallus gallus

Seq: Struc:

Seq: Struc:

Seq: Struc:

Seq: Struc:					1906 a.a. 302 a.a.*

Protein chain

P42212 (GFP_AEQVI) - Green fluorescent protein from Aequorea victoria

Seq: Struc:					238 a.a. 302 a.a.*

Key:

PfamA domain

Secondary structure

CATH domain

* PDB and UniProt seqs differ at 348 residue positions (black crosses)



		Enzyme reactions

Enzyme class:

E.C.2.7.11.18 - [myosin light-chain] kinase.

[IntEnz] [ExPASy] [KEGG] [BRENDA]

Reaction:

1.	L-seryl-[myosin light chain] + ATP = O-phospho-L-seryl-[myosin light chain] + ADP + H⁺
2.	L-threonyl-[myosin light chain] + ATP = O-phospho-L-threonyl-[myosin light chain] + ADP + H⁺

L-seryl-[myosin light chain]	+	ATP	=	O-phospho-L-seryl-[myosin light chain]	+	ADP	+	H(+)

L-threonyl-[myosin light chain]	+	ATP	=	O-phospho-L-threonyl-[myosin light chain]	+	ADP	+	H(+)

Cofactor:

Ca(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M807657200	J Biol Chem 284:6455-6464 (2009)
PubMed id: 19098007

Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design.
J.Akerboom, J.D.Rivera, M.M.Guilbe, E.C.Malavé, H.H.Hernandez, L.Tian, S.A.Hires, J.S.Marvin, L.L.Looger, E.R.Schreiter.

ABSTRACT

The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca2+-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.

Selected figure(s)



	Figure 3. Stereoview of the structures of Ca^2^+-saturated GCaMP2-K387W monomer (red), Ca^2^+-saturated GCaMP2 dimer (yellow), and Ca^2^+-free 8EF-GCaMP2 (blue) superimposed using the cpEGFP domain. The proteins are represented as ribbons with the cpEGFP chromophore represented as sticks.		Figure 5. A rationally designed, improved GCaMP2 variant. A, fluorescence excitation (solid lines) and emission (dashed lines) spectra of Ca^2+-saturated GCaMP2 T116V (red) and T116V/D381Y (blue), as well as their calcium-free forms (gray and black, respectively). Normalized absorbance spectra of each are shown in the inset. B, close-up stereo view of the Ca^2+-saturated monomeric GCaMP2 structure, showing the location of aspartate 381 (D381) of CaM at the CaM/cpEGFP domain interface. GCaMP2 is displayed as ribbons colored by domain. The side chain of Asp^381 and the cpEGFP chromophore are displayed as sticks. A model of a tyrosine side chain at position 381 is shown in semitransparent sticks to represent a possible conformation of the D381Y mutant and to illustrate the proximity of this side chain to the chromophore.

Figures were selected by an automated process.

Literature references that cite this PDB file's key reference

PubMed id

Reference

21251723

A.E.Palmer, Y.Qin, J.G.Park, and J.E.McCombs (2011).
Design and application of genetically encoded biosensors.

Trends Biotechnol, 29, 144-152.

21383146

A.Muto, M.Ohkura, T.Kotani, S.Higashijima, J.Nakai, and K.Kawakami (2011).
Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish.

Proc Natl Acad Sci U S A, 108, 5425-5430.

20010839

A.Kirchhofer, J.Helma, K.Schmidthals, C.Frauer, S.Cui, A.Karcher, M.Pellis, S.Muyldermans, C.S.Casas-Delucchi, M.C.Cardoso, H.Leonhardt, K.P.Hopfner, and U.Rothbauer (2010).
Modulation of protein properties in living cells using nanobodies.

Nat Struct Mol Biol, 17, 133-138.

PDB codes:

3g9a 3k1k

20521333

H.J.Carlson, D.W.Cotton, and R.E.Campbell (2010).
Circularly permuted monomeric red fluorescent proteins with new termini in the beta-sheet.

Protein Sci, 19, 1490-1499.

20944239

N.Suzuki, M.Hiraki, Y.Yamada, N.Matsugaki, N.Igarashi, R.Kato, I.Dikic, D.Drew, S.Iwata, S.Wakatsuki, and M.Kawasaki (2010).
Crystallization of small proteins assisted by green fluorescent protein.

Acta Crystallogr D Biol Crystallogr, 66, 1059-1066.

PDB codes:

3ai4 3ai5

20167470

S.Okumoto (2010).
Imaging approach for monitoring cellular metabolites and ions using genetically encoded biosensors.

Curr Opin Biotechnol, 21, 45-54.

19742327

A.E.Granstedt, M.L.Szpara, B.Kuhn, S.S.Wang, and L.W.Enquist (2009).
Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus.

PLoS One, 4, e6923.

20064437

A.Perron, H.Mutoh, T.Launey, and T.Knöpfel (2009).
Red-shifted voltage-sensitive fluorescent proteins.

Chem Biol, 16, 1268-1277.

19935839

C.D.Wilms, and M.Häusser (2009).
Lighting up neural networks using a new generation of genetically encoded calcium sensors.

Nat Methods, 6, 871-872.

19898485

L.Tian, S.A.Hires, T.Mao, D.Huber, M.E.Chiappe, S.H.Chalasani, L.Petreanu, J.Akerboom, S.A.McKinney, E.R.Schreiter, C.I.Bargmann, V.Jayaraman, K.Svoboda, and L.L.Looger (2009).
Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.

Nat Methods, 6, 875-881.

19771330

W.B.Frommer, M.W.Davidson, and R.E.Campbell (2009).
Genetically encoded biosensors based on engineered fluorescent proteins.

Chem Soc Rev, 38, 2833-2841.

The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.