Crystallization and preliminary x-ray diffraction studies on the conserved GTPase domain of the signal recognition particle from Acidianus ambivalens.
The signal recognition particle (SRP) of bacteria consists of only one protein, known as Ffh or the SRP54 homologue, which forms a complex with 4.5S RNA. It also binds to signal peptides and contains a GTPase which displays interesting differences to Ras GTPases. The conserved NG-domain of Ffh from the archaebacterium Acidianus ambivalens was cloned and overexpressed with a C-terminal His tag in Escherichia coli. Crystallization experiments of the native protein as well as of the Thr112Ala mutant, which is deficient in GTP hydrolysis, resulted in crystals suitable for X-ray diffraction. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 64.5, b = 128.3, c = 72.0 A. At cryogenic temperatures, the crystals diffracted to a resolution limit of 2.8 A using a rotating-anode generator and contain one molecule per asymmetric unit. A native data set has been collected using synchrotron radiation to around 2.0 A resolution. Selenomethionine protein was produced; its crystals diffract in-house to about 2.8 A resolution.
Montoya
G
G
European Molecular Biology Laboratory, Structural Biology Programme, Postfach 10.2209, D--69017 Heidelberg, Germany.
te Kaat
K
K
Moll
R
R
Schäfer
G
G
Sinning
I
I
D001105:Archaea; D019843:Archaeal Proteins / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003460:Crystallization; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012645:Selenomethionine / Q000737:chemistry; D051140:Selenoproteins; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000235:genetics; D017356:Synchrotrons; D014961:X-Ray Diffraction
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D010446:Peptide Fragments; D011506:Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D051140:Selenoproteins; D018271:Signal Recognition Particle; D012645:Selenomethionine; D020558:GTP Phosphohydrolases