Abstract
GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology. The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change. In this study, we determined crystal structures of Ca2+-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its parent version GCaMP5G. Our analyses reveal the structural basis for the outperformance of this newly developed Ca2+ indicator. Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and increased fluorescence intensity common to all three versions of GCaMP6. Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity, maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling. Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Tank DW, Sugimori M, Connor JA, Llinás RR. Spatially resolved calcium dynamics of mammalian Purkinje cells in cerebellar slice. Science, 1988, 242: 773–777
Sabatini BL, Oertner TG, Svoboda K. The life cycle of Ca2+ ions in dendritic spines. Neuron, 2002, 33: 439–452
Kerr R, Lev-Ram V, Baird G, Vincent P, Tsien RY, Schafer WR. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron, 2000, 26: 583–594
Ohki K, Chung S, Ch’ng YH, Kara P, Reid RC. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature, 2005, 433: 597–603
Jia H, Rochefort NL, Chen X, Konnerth A. Dendritic organization of sensory input to cortical neurons in vivo. Nature, 2010, 464: 1307–1312
Huber D, Gutnisky DA, Peron S, O’Connor DH, Wiegert JS, Tian L, Oertner TG, Looger LL, Svoboda K. Multiple dynamic represen-tations in the motor cortex during sensorimotor learning. Nature, 2012, 484: 473–478
Stosiek C, Garaschuk O, Holthoff K, Konnerth A. In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci USA, 2003, 100: 7319–7324
Tour O, Adams SR, Kerr RA, Meijer RM, Sejnowski TJ, Tsien RW, Tsien RY. Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator. Nat Chem Biol, 2007, 3: 423–431
Palmer AE, Tsien RY. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Prot, 2006, 1: 1057–1065
Burgoyne RD. Neuronal calcium sensor proteins: generating diversity in neuronal Ca2+ signalling. Nat Rev Neurosci, 2007, 8: 182–193
Nakai J, Ohkura M, Imoto K. A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat Biotechnol, 2001, 19: 137–141
Wang Q, Shui B, Kotlikoff MI, Sondermann H. Structural basis for calcium sensing by GCaMP2. Structure, 2008, 16: 1817–1827
Akerboom J, Rivera JD, Guilbe MM, Malavé EC, Hernandez HH, Tian L, Hires SA, Marvin JS, Looger LL, Schreiter ER. Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. J Biol Chem, 2009, 284: 6455–6464
4 Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA, Schreiter ER, Bargmann CI, Jayaraman V, Svoboda K, Looger LL. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods, 2009, 6: 875–881
Akerboom J, Chen TW, Wardill TJ, Tian L, Marvin JS, Mutlu S, Calderón NC, Esposti F, Borghuis BG, Sun XR, Gordus A, Orger MB, Portugues R, Engert F, Macklin JJ, Filosa A, Aggarwal A, Kerr RA, Takagi R, Kracun S, Shigetomi E, Khakh BS, Baier H, Lagnado L, Wang SS, Bargmann CI, Kimmel BE, Jayaraman V, Svoboda K, Kim DS, Schreiter ER, Looger LL. Optimization of a GCaMP calcium indicator for neural activity imaging. J Neurosci, 2012, 32: 13819–13840
Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature, 2013, 499: 295–300
Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr, 2011, 67: 271–281
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr, 2011, 67: 235–242
McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ. Likelihood-enhanced fast translation functions. Acta Crystallogr D Biol Crystallogr, 2005, 61: 458–464
Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr, 2012, 68: 352–367
Headd JJ, Echols N, Afonine PV, Grosse-Kunstleve RW, Chen VB, Moriarty NW, Richardson DC, Richardson JS, Adams PD. Use of knowledge-based restraints in phenix.refine to improve macro-molecular refinement at low resolution. Acta Crystallogr D Biol Crystallogr, 2012, 68: 381–390
Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr, 2010, 66: 486–501
Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr, 2010, 66: 12–21
Author information
Authors and Affiliations
Corresponding authors
Additional information
Contributed equally to this work
This article is published with open access at link.springer.com
Rights and permissions
Open Access This article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
About this article
Cite this article
Ding, J., Luo, A.F., Hu, L. et al. Structural basis of the ultrasensitive calcium indicator GCaMP6. Sci. China Life Sci. 57, 269–274 (2014). https://doi.org/10.1007/s11427-013-4599-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11427-013-4599-5