Year: 2011
Type: article
Abstract: Poster Sessions C355reproducible as tetragonal bipyramides.Diffraction images of different crystals show split or smeared spots but symmetry is clearly visible.Indexing works properly using different programs suggesting a primitive tetragonal lattice with a = b = 66.8 Å and c = 113.2Å and one molecule in the asymmetric unit.Merging statistics and automatic space group assignment in various program packages suggests point group 422 with a 4 1/3 screw axis along the fourfold and also a twofold screw axis (space group P4 1 2 1 2 or P4 3 2 1 2).But some strong violations of systematic absences at low resolution along the fourfold and one twofold axis indicate that space group P4 1/3 2 1 2 may not actually be correct.Despite this, a three-wavelength SeMet-MAD dataset was measured with a resolution of 2.8 Å and useable anomalous signal to about 3.5 Å. SHELXD [1] consistently found a solution for the Sesubstructure in space group P4 1 2 1 2 (4 out of 5 Se atoms with CC all of 52.9 and CC weak of 33.6).After solvent flattening with SHELXE [1], the experimental electron density revealed the expected three helical bundle.No improvement was achieved in further attempts of density modification using different programs from the CCP4 program suite [2].Using the Se positions as anchors for sequence assignment, around 80 amino acids (out of a total of 221) could be built, another 40 could be placed with some uncertainty.The free R-factor stalled at ~46 % with a FOM of ~50 %, and little difference density that would have allowed to extend the model.This model was taken for molecular replacement with PHASER [3] into the single datasets of the MAD experiment and other datasets from the native protein.Single solutions were found but with rather low Z-scores of ~3.6 (rotation function) and ~6 (translation function).We also tried molecular replacement with lower symmetry down to P4 1 , P2 1 2 1 2 and C222 1 as well as experimental phasing in these space groups.But this did not result in better phases that allowed extending the structure or refining it to lower R-factors.Apparently, this is a case of severe pseudosymmetry as refinement of an initial model in the suggested high symmetry spacegroup P4 1 2 1 2 did not work.Pseudo-merohedral twinning in a lower symmetry space group seems possible but analysis with PHENIX Xtriage [4] did not detect any indication for this.Experimental phasing in space groups P2 1 or P1 did not work due to lower multiplicity of the data.After all, other crystallographic or biochemical approaches may be required to determine the structure of this protein.
Authors: N. Matsugaki, Y. Yamada, L. M. G. Chavas, M. Hiraki, M. Kawasaki, R. Kato, N. Igarashi, S. Wakatsuki
Language: English
Subfield: Structural Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2326468166