Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium.

Léger C, Pitard I, Sadi M, Carvalho N, Brier S, Mechaly A, Raoux-Barbot D, Davi M, Hoos S, Weber P, Vachette P, Durand D, Haouz A, Guijarro JI, Ladant D, Chenal A, BMC Biol 20(1):176 (2022) Europe PMC

SASDNX3 – Calcium-bound Calmodulin, including structural models

MWexperimental 16 kDa
MWexpected 17 kDa
VPorod 33 nm3
log I(s) 2.02×10-2 2.02×10-3 2.02×10-4 2.02×10-5
Calmodulin-1 small angle scattering data  s, nm-1
ln I(s)
Calmodulin-1 Guinier plot ln 2.03×10-2 Rg: 2.2 nm 0 (2.2 nm)-2 s2
Calmodulin-1 Kratky plot 1.104 0 3 sRg
Calmodulin-1 pair distance distribution function Rg: 2.2 nm 0 Dmax: 7.2 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Calcium-bound Calmodulin, including structural models Rg histogram Rg, nm
Calmodulin-1 EOM/RANCH model
Calmodulin-1 EOM/RANCH model
Calmodulin-1 EOM/RANCH model
Calmodulin-1 EOM/RANCH model

log I(s)
 s, nm-1

Synchrotron SAXS data from solutions of calcium-bound calmodulin in 20 mM Hepes, 150 mM NaCl, 2 mM CaCl2, pH 7.4 were collected on the SWING beam line at the SOLEIL storage ring (Saint-Aubin, France) using a EIGER 4M detector at a sample-detector distance of 2.0 m and at a wavelength of λ = 0.1033 nm (I(s) vs s, where s = 4πsin θ/λ and 2θ is the scattering angle). 1320 successive 1 second frames (0.990 second exposure time / 0.010 second dead time) were collected at 15°C using size-exclusion chromatography SAXS. A 50 μl sample containing 500 µM calmodulin was injected onto a TSKgel G3000SW column (Tosoh Bioscience) and eluted at a 0.50 ml/min flow rate. Scattered intensities were converted into absolute scale (cm-1) values using the scattering of water. Molecular mass was estimated at 15.6kDa using the correlation volume Vc (Rambo, R.P. and Tainer, J. A. (2013) Nature 496(7446):477-81.)

Ab initio model and modelling using an ensemble of conformations: Bottom panel: Comparison of the experimental data (blue dots) with the calculated scattering pattern (red line) of the DENSS density distribution shown on the right. (Grant, T. D. (2018) Nat Methods 15, 191–193). Top panel: Comparison of the experimental data (blue dots) with the calculated scattering pattern (red line) of the best EOM ensemble models shown on the right. Chi2 = 1.06. The program Ranch within EOM creates a large (10,000) pool of random conformations, starting from the structures of the two calcium-bound lobes of holo-CaM (PDB ID: 1CLL). Amino acids (76 – 81) in the interlobe linker region were described as dummy residues. We used the native option to generate the linker separating the two lobes of CaM. Dummy residues are subsequently substituted by complete residues using the programs PD2 (Moore, B. L., Kelley, L. A., Barber, J., Murray, J. W., and MacDonald, J. T. (2013) J Comput Chem 34, 1881–9) and SCWRL4 (Krivov, G. G., Shapovalov, M. V., and Dunbrack, R. L. (2009) Proteins 77, 778–95.) before calculating all scattering patterns using CRYSOL. The routine Gajoe within EOM tries to fit the experimental scattering curve by the average of the calculated scattering patterns of an ensemble of conformations using a genetic algorithm protocol. (Tria, G., Mertens, H. D. T., Kachala, M., and Svergun, D. I. (2015) IUCrJ 2, 207-217.)

Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   16.7 kDa
UniProt   P0DP23 (2-149)
Sequence   FASTA