Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.

Fuertes G, Banterle N, Ruff KM, Chowdhury A, Mercadante D, Koehler C, Kachala M, Estrada Girona G, Milles S, Mishra A, Onck PR, Gräter F, Esteban-Martín S, Pappu RV, Svergun DI, Lemke EA, Proc Natl Acad Sci U S A 114(31):E6342-E6351 (2017) Europe PMC

SASDEJ2 – Labeled nucleoporin NUP49/NSP49 (N49-Alexa488/Alexa594) without denaturant

Nucleoporin NUP49/NSP49
Alexa Fluor™ 594 C5 Maleimide
Alexa Fluor™ 488 C5 Hydroxylamine
MWexperimental 7 kDa
MWexpected 5 kDa
VPorod 12 nm3
log I(s) 2.45×102 2.45×101 2.45×100 2.45×10-1
Nucleoporin NUP49/NSP49 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine small angle scattering data  s, nm-1
ln I(s)
Nucleoporin NUP49/NSP49 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine Guinier plot ln 2.46×102 Rg: 1.9 nm 0 (1.9 nm)-2 s2
(sRg)2I(s)/I(0)
Nucleoporin NUP49/NSP49 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine Kratky plot 1.104 0 3 sRg
p(r)
Nucleoporin NUP49/NSP49 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine pair distance distribution function Rg: 1.9 nm 0 Dmax: 6.5 nm

Data validation


There are no models related to this curve.

Synchrotron SAXS data from solutions of Labeled nucleoporin NUP49/NSP49 (N49-Alexa488/Alexa594) without denaturant in PBS, 10 mM DTT, pH 7.4 were collected on the EMBL P12 beam line at the PETRA III storage ring (Hamburg, Germany) using a Pilatus 2M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.1 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 2 and 4 mg/ml were measured at 23°C. 20 successive 0.050 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted. The low angle data collected at lower concentration were merged with the highest concentration high angle data to yield the final composite scattering curve.

The protein contains a penultimate non-canonical amino acid p-acetylphenylalanine (207 Da) that is represented as U (selenocysteine, 168 Da) in the amino acid sequence for the entry. Therefore, the calculated MW from sequence (MW(expected)) must be adjusted accordingly (ca. 40 Da).

Tags: idp
Nucleoporin NUP49/NSP49 (N49-488/594)
Mol. type   Protein
Organism   Saccharomyces cerevisiae
Olig. state   Monomer
Mon. MW   3.9 kDa
 
UniProt   Q02199 (121-154)
Sequence   FASTA
 
Alexa Fluor™ 594 C5 Maleimide (Alexa594)
Mol. type   Other
Olig. state   Monomer
Mon. MW   0.9 kDa
Alexa Fluor™ 488 C5 Hydroxylamine (Alexa488)
Mol. type   Other
Olig. state   Monomer
Mon. MW   0.7 kDa