PDBsum entry 3c0v

Plant protein

PDB id: 3c0v

Contents

Protein chains

151 a.a. *

Ligands

ZEA ×6

TBR ×5

EPE

Metals

_NA ×2

Waters ×578

* Residue conservation analysis

PDB id:

3c0v

Name:

Plant protein

Title:

Crystal structure of cytokinin-specific binding protein in complex with cytokinin and ta6br12

Structure:

Cytokinin-specific binding protein. Chain: a, b, c, d. Engineered: yes

Source:

Vigna radiata. Gene: vrcsbp. Expressed in: escherichia coli.

Resolution:

1.80Å R-factor: 0.159 R-free: 0.207

Authors:

O.Pasternak,A.Bujacz,J.Biesiadka,G.Bujacz,M.Sikorski,M.Jaskolski

Key ref:

O.Pasternak et al. (2008). MAD phasing using the (Ta6Br12)2+ cluster: a retrospective study. Acta Crystallogr D Biol Crystallogr, 64, 595-606. PubMed id: 18453695 DOI: 10.1107/S0907444908007853

Date:

21-Jan-08 Release date: 20-May-08

Protein chains

A0A1S3THR8  (A0A1S3THR8_VIGRR) -  Phytohormone-binding protein CSBP from Vigna radiata var. radiata

Seq: 155 a.a.

Struc: 151 a.a.

DOI no: 10.1107/S0907444908007853

Acta Crystallogr D Biol Crystallogr 64:595-606 (2008)

PubMed id: 18453695

MAD phasing using the (Ta6Br12)2+ cluster: a retrospective study.

O.Pasternak, A.Bujacz, J.Biesiadka, G.Bujacz, M.Sikorski, M.Jaskolski.

ABSTRACT

The crystal structure of cytokinin-specific binding protein (CSBP) containing four independent molecules with 4 x 155 = 620 residues in the asymmetric unit of the P6(4) unit cell has been solved by three-wavelength MAD using 1.8 angstroms resolution data recorded from a crystal derivatized with the dodecabromohexatantalum cation (Ta6Br12)2+. The diffraction data contained a very strong anomalous signal (allowing successful phasing even using peak SAD data alone) despite the fact that the five (Ta6Br12)2+ clusters found in the asymmetric unit have low occupancy (about 0.3). The derivative structure has been successfully refined to R = 0.158, providing interesting details on the geometry of the (Ta6Br12)2+ cluster, its interactions with the protein and on the backsoaking of a cytokinin ligand that was originally part of a CSBP-cytokinin complex in the native crystals used for (Ta6Br12)2+ derivatization. A simulation analysis of the phasing power of the (Ta6Br12)2+ ions at artificially imposed resolution limits shows that it is not possible to resolve the individual Ta atoms if the dmin limit of the data is higher than 2.9 angstroms. Additionally, for successful Ta identification the (Ta6Br12)2+ complex should be specifically bound and ordered. Good binding at the protein surface is facilitated by the presence of acidic groups, indicating higher pH buffer conditions to be preferable. In addition, the water channels in the crystal should be sufficiently wide (at least 11 angstroms) to allow free diffusion of the (Ta6Br12)2+ ions on soaking. A retrospective look at the initial molecular-replacement calculations provides interesting insights into how the peculiar packing mode and strong bias of the molecular-replacement-phased electron-density maps had hindered successful solution of the structure by this method.

Selected figure(s)

Figure 4.
Figure 4 Anomalous difference map calculated for the best Ta[6]Br[12] cluster, TBR1, using phases generated by SOLVE and subsequent density modification for Ta-MAD data truncated at different d[min] levels: 1.8 Å (a), 2.6 Å (b) and 2.9 Å (c). The maps were contoured at levels of 18 for (a) and (c) or 6 for (b). (b) must be contoured at a lower level to emphasize its features or it would appear to be spherical. In (c), lowering of the contour level does not reveal any real features. The dark balls indicate the Ta positions located automatically by SOLVE.

Figure 6.
Figure 6 (Ta[6]Br[12])^2+-binding sites. The four CSBP molecules arranged as in the crystallographic asymmetric unit are coloured green (A), blue (B), red (C) and yellow (D). The (Ta[6]Br[12])^2+ clusters are marked using orange Ta spheres and violet Br spheres. (a) An overview of the relation between the four CSBP molecules and the five (Ta[6]Br[12])^2+ ions. The symmetry-related protein molecules that complete the (Ta[6]Br[12])^2+-binding sites have been omitted for clarity. (b) Close-up view of the binding details of two representative (Ta[6]Br[12])^2+ ions. The binding site of the TBR2 ion is shown on the left. The same interactions are observed for the TBR1 binding site. The binding site of the TBR4 ion is shown on the right. A similar environment is observed for TBR5. The amino-acid residues located in the vicinity of the (Ta[6]Br[12])^2+ ions are shown in stick representation.

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2008, 64, 595-606) copyright 2008.

Figures were selected by the author.