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