Expression and analysis of the SAM-dependent RNA methyltransferase Rsm22 from Saccharomyces cerevisiae

Alam J, Rahman F, Sah-Teli S, Venkatesan R, Koski M, Autio K, Hiltunen J, Kastaniotis A, Acta Crystallographica Section D Structural Biology 77(6) (2021) DOI

SASDJT5 – Probable S-adenosyl-L-methionine-dependent RNA methyltransferase RSM22, mitochondrial-dimer

dimeric Probable S-adenosyl-L-methionine-dependent RNA methyltransferase RSM22, mitochondrial
MWexperimental 220 kDa
MWexpected 141 kDa
VPorod 516 nm3
log I(s) 1.07×10-1 1.07×10-2 1.07×10-3 1.07×10-4
dimeric Probable S-adenosyl-L-methionine-dependent RNA methyltransferase RSM22, mitochondrial small angle scattering data  s, nm-1
ln I(s)
dimeric Probable S-adenosyl-L-methionine-dependent RNA methyltransferase RSM22, mitochondrial Guinier plot ln 1.07×10-1 Rg: 5.0 nm 0 (5.0 nm)-2 s2
(sRg)2I(s)/I(0)
dimeric Probable S-adenosyl-L-methionine-dependent RNA methyltransferase RSM22, mitochondrial Kratky plot 1.104 0 √3 sRg
p(r)
dimeric Probable S-adenosyl-L-methionine-dependent RNA methyltransferase RSM22, mitochondrial pair distance distribution function Rg: 5.0 nm 0 Dmax: 18.2 nm

Data validation

Fits and models

log I(s)
 s, nm-1
dimeric Probable S-adenosyl-L-methionine-dependent RNA methyltransferase RSM22, mitochondrial DAMMIN model
Rsm22 was purified in 40mM Tris pH7.5, 500mM NaCl, 5% Glycerol, 2.5mM DTT for SAXS purpose. Batch mode SAXS studies were carried out on the purified Rsm22 at B21 beamline of Diamond Light Source (DLS), UK. 40 µL of Rsm22 was used and 120 frames were collected. Scattered X-rays at a wavelength of 0.1 nm (at 12.4 keV) were recorded with a Pilatus 2M detector. The buffer scatterings were subtracted from protein scatterings using the program SCATTER (Reference: Förster, S., Apostol, L. and Bras, W. (2010)) and PRIMUS (Reference: Konarev, P. V., (2003)). The radius of gyration Rg, forward scattering I0, the maximum dimensions Dmax and the interatomic distance distribution functions P(r) were estimated using the GNOM package (Reference: Svergun, D. I. (1992)) in SCATTER and PRIMUS. The ab initio models of Rsm22 were generated by DAMMIN of the online-SAXS cluster at EMBL, Hamburg (Reference: D. I. Svergun (1999)) using the output file from GNOM package.

dimeric Probable S-adenosyl-L-methionine-dependent RNA methyltransferase RSM22, mitochondrial (Yeast Rsm22-dimer)
Mol. type   Protein
Organism   Saccharomyces cerevisiae
Olig. state   Dimer
Mon. MW   70.3 kDa
 
UniProt   P36056 (17-628)
Sequence   FASTA