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FP5

REPBIOTECH Résumé de rapport

Project ID: QLK3-CT-2002-02071
Financé au titre de: FP5-LIFE QUALITY
Pays: Sweden

3 D structure of archaeal MCM and nuclease

- The attempts to solve the structure of the P. abysssi RF-C were unsuccessful due to high instability of the complex. Moreover, the human RF-C structure was solved by other research groups during the course of the project, reducing considerably the priority on this specific objective.

- The structure of the N-terminal part of SsoMCM was determined by molecular replacement using the available homologous structure from Methanobacterium thermoautotrophicum as the search model. A partial solution was obtained for the hexagonal crystal form with the program PHASER. Only three of the four subunits in the asymmetric unit suggested by Matthews crystal packing probability calculation (Kantardjieff and Rupp 2003) could be detected. For the monoclinic crystal form, a complete solution was achieved with three subunits in the AU. Interestingly, by application of the crystallographic symmetry operators to the trimer we were able to build up the ring-shaped hexameric structure.

The DNA complex of the N-terminal part has been solved. The manuscript is under completion.

- DNA nuclease Pab 2263: This protein was identified as an interesting target as a result of the search of novel interacting partners. Pab2263 interacts with PCNA and was recently found to have nuclease activity. The first native protein batch was purified and then crystallized by the sitting drop vapour diffusion method. The crystals belonged to the orthorhombic system and space group C2221 with cell dimensions of a = 78.2 Å, b = 101.1 Å, c = 155.9 Å, a= b =g = 90°. It was found that there were two molecules in the asymmetric unit with a local 2-fold axis at the relative coordinates x,z = (0.0125, 0.2268), which is parallel to crystallographic 21 axis.

An initial diffraction data set was collected at DESY, Hamburg, Germany. Since there is no similar structure available, which could be used in a molecular replacement search, we have chosen the multiple-wavelength anomalous diffraction (MAD) method to solve the structure of Pab2263. A Seleno-methionine substituted derivative protein was expressed and purified. The derivative protein was crystallized in the same space group under similar crystallization conditions as the native protein but with different cell dimensions of a = 81.4 Å, b = 159.8 Å, c = 100.8 Å, a= b = g= 90°. A MAD data set was collected at DESY.

Data analysis revealed that only two of the three methionines were substituted, which gave a relatively weak anomalous diffraction signal in particular for the high resolution X-ray data. Anyway a medium resolution electron density map (at Ü 3.5 Å resolution) could be calculated with the available phase information from the MAD data. The density features of this map suggested that Pab2263 forms a complex ring-like structure.

Informations connexes

Contact

Rudolf LADENSTEIN, (Director of laboratory)
Tél.: +46-8-6089222
Fax: +46-8-6089290
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