9K8W

Crystal structure of the calcium indicator GCaMP6s-BrUS in calcium-bound state

Summary for 9K8W

• Entry DOI: 10.2210/pdb9k8w/pdb
• Descriptor: Calcium indicator GCaMP6s-BrUS,Calmodulin-1, CALCIUM ION (3 entities in total)
• Functional Keywords: genetically encoded calcium indicator, calcium-bound state, calcium-sensitive fluorescence lifetime, calcium saturated, fluorescent protein
• Biological source: synthetic construct; More
• Total number of polymer chains: 1
• Total formula weight: 52192.37

Authors

Varfolomeeva, L.A.,Simonyan, T.R.,Mamontova, A.V.,Bogdanov, A.M.,Popov, V.O.,Boyko, K.M. (deposition date: 2024-10-24, release date: 2024-12-11, Last modification date: 2025-01-01)

Primary citation

Simonyan, T.R.,Varfolomeeva, L.A.,Mamontova, A.V.,Kotlobay, A.A.,Gorokhovatsky, A.Y.,Bogdanov, A.M.,Boyko, K.M.
Calcium Indicators with Fluorescence Lifetime-Based Signal Readout: A Structure-Function Study.
Int J Mol Sci, 25:-, 2024

PubMed Abstract: The calcium cation is a crucial signaling molecule involved in numerous cellular pathways. Beyond its role as a messenger or modulator in intracellular cascades, calcium's function in excitable cells, including nerve impulse transmission, is remarkable. The central role of calcium in nervous activity has driven the rapid development of fluorescent techniques for monitoring this cation in living cells. Specifically, genetically encoded calcium indicators (GECIs) are the most in-demand molecular tools in their class. In this work, we address two issues of calcium imaging by designing indicators based on the successful GCaMP6 backbone and the fluorescent protein BrUSLEE. The first indicator variant (GCaMP6s-BrUS), with a reduced, calcium-insensitive fluorescence lifetime, has potential in monitoring calcium dynamics with a high temporal resolution in combination with advanced microscopy techniques, such as light beads microscopy, where the fluorescence lifetime limits acquisition speed. Conversely, the second variant (GCaMP6s-BrUS-145), with a flexible, calcium-sensitive fluorescence lifetime, is relevant for static measurements, particularly for determining absolute calcium concentration values using fluorescence lifetime imaging microscopy (FLIM). To identify the structural determinants of calcium sensitivity in these indicator variants, we determine their spatial structures. A comparative structural analysis allowed the optimization of the GCaMP6s-BrUS construct, resulting in an indicator variant combining calcium-sensitive behavior in the time domain and enhanced molecular brightness. Our data may serve as a starting point for further engineering efforts towards improved GECI variants with fine-tuned fluorescence lifetimes.

PubMed: 39684209
DOI: 10.3390/ijms252312493

Experimental method

X-RAY DIFFRACTION (2.65 Å)