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LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae.

Kusakizako T, Tanaka Y, Hipolito CJ, Kuroda T, Ishitani R, Suga H, Nureki O - Acta Crystallogr F Struct Biol Commun (2016)

Bottom Line: Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane.Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets.Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

ABSTRACT
Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.

No MeSH data available.


Related in: MedlinePlus

X-ray diffraction images of the form A (a) and form B (b) crystals. The rings indicate 2.5 Å (a) and 2.2 Å (b) resolution, respectively.
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fig3: X-ray diffraction images of the form A (a) and form B (b) crystals. The rings indicate 2.5 Å (a) and 2.2 Å (b) resolution, respectively.

Mentions: A form A single crystal diffracted X-rays to 2.5 Å resolution (Fig. 3 ▸a) and belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. The calculated Matthews coefficient is 2.58 Å3 Da−1 with 52.4% solvent content, assuming the presence of one molecule in the asymmetric unit. The form B single crystal diffracted X-rays to 2.2 Å resolution (Fig. 3 ▸b) and belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The calculated Matthews coefficient is 3.01 Å3 Da−1 with 59.2% solvent content, assuming the presence of one molecule in the asymmetric unit. Molecular replacement was performed using the structure of PfMATE as the search model and an interpretable electron-density map was obtained. However, we have not yet observed an electron-density peak corresponding to either the macrocyclic peptide or Hoechst 33342. Model building and structure refinement are under way.


LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae.

Kusakizako T, Tanaka Y, Hipolito CJ, Kuroda T, Ishitani R, Suga H, Nureki O - Acta Crystallogr F Struct Biol Commun (2016)

X-ray diffraction images of the form A (a) and form B (b) crystals. The rings indicate 2.5 Å (a) and 2.2 Å (b) resolution, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4933005&req=5

fig3: X-ray diffraction images of the form A (a) and form B (b) crystals. The rings indicate 2.5 Å (a) and 2.2 Å (b) resolution, respectively.
Mentions: A form A single crystal diffracted X-rays to 2.5 Å resolution (Fig. 3 ▸a) and belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. The calculated Matthews coefficient is 2.58 Å3 Da−1 with 52.4% solvent content, assuming the presence of one molecule in the asymmetric unit. The form B single crystal diffracted X-rays to 2.2 Å resolution (Fig. 3 ▸b) and belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The calculated Matthews coefficient is 3.01 Å3 Da−1 with 59.2% solvent content, assuming the presence of one molecule in the asymmetric unit. Molecular replacement was performed using the structure of PfMATE as the search model and an interpretable electron-density map was obtained. However, we have not yet observed an electron-density peak corresponding to either the macrocyclic peptide or Hoechst 33342. Model building and structure refinement are under way.

Bottom Line: Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane.Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets.Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

ABSTRACT
Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.

No MeSH data available.


Related in: MedlinePlus