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

SASDRX8 – Heterodimeric complex of AfAgo protein Af1318 and AfAgo-N protein (containing N-L1-L2 domains) with a 14 base pair DNA oligoduplex in low salt buffer

Piwi protein AF_1318 (Archaeoglobus fulgidus AfAgo protein)
Uncharacterized protein (AfAgo-N protein containing N-L1-L2 domains)
5'-end phosphorylated DNA oligoduplex, 14 bp (MZ1288)
MWI(0) 78 kDa
MWexpected 90 kDa
VPorod 145 nm3
log I(s) 5.55×10-2 5.55×10-3 5.55×10-4 5.55×10-5
Piwi protein AF_1318 (Archaeoglobus fulgidus AfAgo protein) Uncharacterized protein (AfAgo-N protein containing N-L1-L2 domains) 5'-end phosphorylated DNA oligoduplex, 14 bp (MZ1288) small angle scattering data  s, nm-1
ln I(s)
Piwi protein AF_1318 (Archaeoglobus fulgidus AfAgo protein) Uncharacterized protein (AfAgo-N protein containing N-L1-L2 domains) 5'-end phosphorylated DNA oligoduplex, 14 bp (MZ1288) Guinier plot ln 5.55×10-2 Rg: 2.9 nm 0 (2.9 nm)-2 s2
(sRg)2I(s)/I(0)
Piwi protein AF_1318 (Archaeoglobus fulgidus AfAgo protein) Uncharacterized protein (AfAgo-N protein containing N-L1-L2 domains) 5'-end phosphorylated DNA oligoduplex, 14 bp (MZ1288) Kratky plot 1.104 0 √3 sRg
p(r)
Piwi protein AF_1318 (Archaeoglobus fulgidus AfAgo protein) Uncharacterized protein (AfAgo-N protein containing N-L1-L2 domains) 5'-end phosphorylated DNA oligoduplex, 14 bp (MZ1288) pair distance distribution function Rg: 2.9 nm 0 Dmax: 9.7 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Piwi protein AF_1318 (Archaeoglobus fulgidus AfAgo protein) Uncharacterized protein (AfAgo-N protein containing N-L1-L2 domains) 5'-end phosphorylated DNA oligoduplex, 14 bp (MZ1288) CUSTOM IN-HOUSE model
log I(s)
 s, nm-1
Piwi protein AF_1318 (Archaeoglobus fulgidus AfAgo protein) Uncharacterized protein (AfAgo-N protein containing N-L1-L2 domains) 5'-end phosphorylated DNA oligoduplex, 14 bp (MZ1288) CUSTOM IN-HOUSE model
log I(s)
 s, nm-1
Piwi protein AF_1318 (Archaeoglobus fulgidus AfAgo protein) Uncharacterized protein (AfAgo-N protein containing N-L1-L2 domains) 5'-end phosphorylated DNA oligoduplex, 14 bp (MZ1288) GASBOR model
Synchrotron SAXS data from solutions of the AfAgo and AfAgo-N heterodimer complexed with a 14 base pair DNA oligoduplex in 20 mM TrisHCl pH 7.5, 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 6M 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). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 100.00 μl sample at 14 mg/ml was injected at a 0.50 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column at 20°C. 33 successive 0.995 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.

AfAgo was mixed with dsDNA and AfAgo-N protein in equimolar quantities. The sample was transferred into the corresponding buffer using an NAP Illustra column and concentrated to 14 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.

Piwi protein AF_1318 (Archaeoglobus fulgidus AfAgo protein) (AfAgo)
Mol. type   Protein
Organism   Archaeoglobus fulgidus (strain ATCC 49558 / DSM 4304 / JCM 9628 / NBRC 100126 / VC-16)
Olig. state   Monomer
Mon. MW   50.9 kDa
 
UniProt   O28951 (1-427)
Sequence   FASTA
 
Uncharacterized protein (AfAgo-N protein containing N-L1-L2 domains) (AfAgo-N)
Mol. type   Protein
Organism   Archaeoglobus fulgidus DSM 8774
Olig. state   Monomer
Mon. MW   30.5 kDa
 
UniProt   A0A075WKW4 (1-250)
Sequence   FASTA
 
5'-end phosphorylated DNA oligoduplex, 14 bp (MZ1288) (MZ1288)
Mol. type   DNA
Olig. state   Monomer
Mon. MW   8.7 kDa
Sequence   FASTA