The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein.

Manakova E, Golovinas E, Pocevičiūtė R, Sasnauskas G, Silanskas A, Rutkauskas D, Jankunec M, Zagorskaitė E, Jurgelaitis E, Grybauskas A, Venclovas Č, Zaremba M, Nucleic Acids Res (2024) Europe PMC

SASDRZ8 – Single-chain Archaeoglobus fulgidus argonaute fAfAgo-fusion variant complexed with an 11 base pair DNA oligoduplex in low salt buffer

single-chain full Archaeoglobus fulgidus Argonaute
5'-end phosphorylated DNA guide strand, 11 nt (MZ864)
DNA target strand, 11 nt (MZ865)
MWI(0) 72 kDa
MWexpected 85 kDa
VPorod 125 nm3
log I(s) 5.56×10-2 5.56×10-3 5.56×10-4 5.56×10-5
single-chain full Archaeoglobus fulgidus Argonaute 5'-end phosphorylated DNA guide strand, 11 nt (MZ864) DNA target strand, 11 nt (MZ865) small angle scattering data  s, nm-1
ln I(s)
single-chain full Archaeoglobus fulgidus Argonaute 5'-end phosphorylated DNA guide strand, 11 nt (MZ864) DNA target strand, 11 nt (MZ865) Guinier plot ln 5.56×10-2 Rg: 2.9 nm 0 (2.9 nm)-2 s2
(sRg)2I(s)/I(0)
single-chain full Archaeoglobus fulgidus Argonaute 5'-end phosphorylated DNA guide strand, 11 nt (MZ864) DNA target strand, 11 nt (MZ865) Kratky plot 1.104 0 3 sRg
p(r)
single-chain full Archaeoglobus fulgidus Argonaute 5'-end phosphorylated DNA guide strand, 11 nt (MZ864) DNA target strand, 11 nt (MZ865) pair distance distribution function Rg: 2.9 nm 0 Dmax: 9.6 nm

Data validation


Fits and models


log I(s)
 s, nm-1
single-chain full Archaeoglobus fulgidus Argonaute 5'-end phosphorylated DNA guide strand, 11 nt (MZ864) DNA target strand, 11 nt (MZ865) CUSTOM IN-HOUSE model

log I(s)
 s, nm-1
single-chain full Archaeoglobus fulgidus Argonaute 5'-end phosphorylated DNA guide strand, 11 nt (MZ864) DNA target strand, 11 nt (MZ865) CUSTOM IN-HOUSE model

log I(s)
 s, nm-1
single-chain full Archaeoglobus fulgidus Argonaute 5'-end phosphorylated DNA guide strand, 11 nt (MZ864) DNA target strand, 11 nt (MZ865) DAMMIN model

Synchrotron SAXS data from solutions of a fAfAgo fusion protein complexed with an 11 base pair DNA oligoduplex in 20 mM TrisHCl pH7.5, 200 mM NaCl, 5 mM MgCl2, 1 mM DTT, were collected on the EMBL P12 beam line at PETRA III (DESY, Hamburg, Germany) using a Pilatus 2M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.124 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 3.80 mg/ml was measured at 20°C. 20 successive 0.045 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.

DNA oligoduplex was mixed with scfAfAgo in the presence of MgCl2, buffer was changed using NAP Illustra column and concentrated to 3.8 mg/ml. Note: The DNA sequence of the model shown in this entry is different to the DNA sequence of the oligoduplex in the sample used for SAXS.

single-chain full Archaeoglobus fulgidus Argonaute (sgfAfAgo)
Mol. type   Protein
Organism   Archaeoglobus fulgidus
Olig. state   Monomer
Mon. MW   77.9 kDa
Sequence   FASTA
 
5'-end phosphorylated DNA guide strand, 11 nt (MZ864) (5'p-MZ864)
Mol. type   DNA
Olig. state   Monomer
Mon. MW   3.5 kDa
Sequence   FASTA
 
DNA target strand, 11 nt (MZ865) (MZ865)
Mol. type   DNA
Olig. state   Monomer
Mon. MW   3.4 kDa
Sequence   FASTA