PDBsum entry 2ca6

Signaling regulator

PDB id: 2ca6

Contents

Protein chains

344 a.a.

Ligands

SO4 ×4

Waters ×273

References listed in PDB file

Key reference

Title

Detecting and overcoming hemihedral twinning during the mir structure determination of RNA1p.

Authors

R.C.Hillig, L.Renault.

Ref.

Acta Crystallogr D Biol Crystallogr, 2006, 62, 750-765. [DOI no: 10.1107/S0907444906016222]

PubMed id

16790931

Abstract

The structure of Rna1p was originally solved to 2.7 A resolution by MIRAS from crystals with partial hemihedral twinning in space group I4(1) [Hillig et al. (1999), Mol. Cell, 3, 781-791] by finding a low-twinned native crystal (twin fraction alpha=0.06) and after twin correction of all data sets. Rna1p crystals have now been used to examine how far twinning and twin correction affect MIR phasing with a higher resolution but highly twinned native data set. Even high hemihedral twinning [alphanative=0.39, alphaderivative=0.24] would not have hindered heavy-atom site identification of strong derivatives using difference Patterson maps. However, a weaker derivative could have been missed and refinement would have stalled at high R values had twinning not been identified and accounted for. Twin correction improved both site identification, experimental phasing statistics and MIR map quality. Different strategies were tested for refinement against twinned data. Using uncorrected twinned data and TWIN-CNS, Rna1p has now been refined to 2.2 A resolution (final twinned R and Rfree were 0.165 and 0.218, respectively). The increased resolution enabled release of the NCS restraints and allowed new conclusions to be drawn on the flexibility of the two molecules in the asymmetric unit. In the case of Rna1p, twinned crystal growth was possible owing to the presence of a twofold NCS axis almost parallel to the twin operator.

Figure 1.
Figure 1 Crystals and diffraction pattern of S. pombe Rna1p. (a) Typical sea-urchin-like crystal clusters. (b) Manually separated crystals of about 600 × 40 × 40 µm. (c) Diffraction pattern from native-A (high-resolution sweep, = 0.5°, exposure time 8 min, detector edge 2 Å). Yellow boxes denote the enlarged regions. The reflections show no signs of splitting.

Figure 6.
Figure 6 Structure of Rna1p refined to 2.20 Å. (a) Representative view of the final 3F[o] - 2F[c] electron-density map. Shown is the region around leucine-rich repeat 8 (LRR8), contoured at 1.5 . (b) Ribbon representation of Rna1p. (c) Superimposition of the two independent molecules A (red) and B (blue) in the asymmetric unit (C^ backbone representation). Differences are found in the N-terminal region as well as in LRR3/LRR4. (d) Superimposition of Rna1p (molecules A and B in red and blue, respectively) and the complex of Rna1p (green) with Ran-GMPPNP-Mg-RanBP1 (PDB code 1k5d ). The Ran backbone is shown as a grey ribbon, GMPPNP in stick representation and Mg as magenta-coloured sphere. An enlargement of the region of the flexible loops of LRR3/LRR4 is shown. This region, which differs between molecules A and B in the high-resolution structure of Rna1p, coincides with part of the interface between Rna1p and Ran-GMPPNP. The flexibility in Rna1p may indicate an inherent mobility designed to allow an induced fit.

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2006, 62, 750-765) copyright 2006.

Secondary reference #1

Title

The crystal structure of RNA1p: a new fold for a gtpase-Activating protein.

Authors

R.C.Hillig, L.Renault, I.R.Vetter, T.Drell, A.Wittinghofer, J.Becker.

Ref.

Mol Cell, 1999, 3, 781-791. [DOI no: 10.1016/S1097-2765(01)80010-1]

PubMed id

10394366

Figure 2.
Figure 2. NCS-Averaged Experimental Electron Density MapPart of the map showing the region of LRR8 (residues 217–243). The refined model (ball-and-stick) is superimposed on the experimental electron density map after 2-fold NCS averaging, solvent flattening, and histogram matching, contoured at 1.0 σ. Figure prepared using Bobscript ([28]).

Figure 4.
Figure 4. Structural Overlay of the 11 LRRs of rna1pLRRs that deviate markedly from the ideal LRR structure are presented in blue (LRR1), red (LRR3), and green (LRR5). The position of Arg-74 is marked by a red ball. The LRR consensus residues of LRR8 are depicted as ball-and-stick models (yellow) illustrating the formation of the hydrophobic core. Figure produced with Bobscript ([28]).

The above figures are reproduced from the cited reference with permission from Cell Press

Secondary reference #2

Title

Rangap mediates gtp hydrolysis without an arginine finger.

Authors

M.J.Seewald, C.Körner, A.Wittinghofer, I.R.Vetter.

Ref.

Nature, 2002, 415, 662-666. [DOI no: 10.1038/415662a]

PubMed id

11832950

Figure 2.
Figure 2: Details of the Ran–RanGAP interface. a, Worm plot of Ran and RanGAP, with colours as in Fig. 1 and important residues shown in ball and stick representation. The uncomplexed RanGAP (cyan) is superimposed; arrows indicate the movement of Lys 76 and Arg 74, and asterisk symbolizes the potential clash of Leu 43 and Lys 76 on complex formation. b, Switch II of Ran (green) superimposed on the Ran–ranBD1 structure (blue), illustrating the reorientation of Gln 69 into a catalytically competent conformation by the residues Tyr 39 from Ran and Asn 131 fron RanGAP.

Figure 3.
Figure 3: The active site. a, The nucleotide and relevant residues of Ran and RanGAP; interactions are indicated by dashed lines. The catalytic water ('W') has very weak density (probably owing to the limited resolution) and was not included in the final model, but is shown here to illustrate its potential interaction partners. It sits in a similar position as in the Ras–RasGAP complex. Asterisk denotes the hydroxyl group of Thr 42 of Ran. The rest of the side chain is omitted for clarity. b, The 2F[o] - F[c] electron density map contoured at 1.6 for the structure with GDP and aluminium fluoride, the latter modelled as AlF[3].

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd