P.L.D. are most abundant. To gain insight into the overall architecture of the ~120 tandem RII domains, we set out to produce, crystallize and determine the 3-D structure of a RII segment spanning four tandem repeats. Here we report the 1.8 ?-resolution crystal structure of the RII tetra-tandemer. It shows how the four RII repeats fold into a rigid and elongated structure in the presence of Ca2+. We used SAXS (small-angle X-ray scattering) to demonstrate the RII tetra-tandemer (four tandem RII) is significantly rigidified in the presence of Ca2+, and that its solution structure is in excellent agreement with the crystal structure. Using a combination of CD, size-exclusion chromatography and AUC (analytical ultracentrifugation) we show Ca2+ is indispensable for folding and rigidifying the structure of the tandem RII domains. We suggest the Ca2+-induced rigidity in the large repetitive extender domains of RTX adhesins is a general mechanism used by Gram-negative Protirelin bacteria, including pathogens, to bind to their specific substrates. MATERIALS AND METHODS Construct design and cloning of the RII tetra-tandemer gene The DNA construct of the RII tetra-tandemer was synthesized by GeneArt (Life Technologies). The four tandem 312-bp repeats were codon-optimized for expression using codon degeneracy while making each repeat as distinct as possible at the DNA sequence level to lessen the chances of recombination (Figure 1). No changes were made to the original aa sequence. Additionally, the GCC content of the DNA sequence was optimized to minimize the formation of RNA secondary structure that could hamper translation. The construct was inserted between BL21DE3 (star) expression cell line. A 1-L culture was grown in the presence of 100?g/ml kanamycin at 37C with shaking until the is the scattering angle. Three Protirelin sample-to-detector distances of 113, 713 and 1513?mm were used to cover an angular range of 0.006 values and elevated concentrations. The normalized background scattering profile of the buffer and polycarbonate cell was subtracted from the normalized sample scattering profiles to obtain the protein scattering curve. The absolute scale calibration of the scattering curves was verified using the known scattering cross-section per unit sample volume, d/d, of water, being d/d (0)=0.01632 cm?1 for molecular shape of the protein in solution was reconstructed using simulated annealing methods implemented in DAMMIN . First, an inverse Fourier transformation was applied to the experimental scattering data to obtain the RDF (radial distribution function), describing the probability of finding interatomic vectors of length (and adjusted to Cav2 give the best fit to the experimental data. The RDF was considered to be zero at that could lead to deletions within the tandem repeats . To circumvent problems with amplification by PCR the gene was synthesized. To avoid recombination the DNA sequence of four identical repeats was altered through codon degeneracy to produce four domains in tandem that, while maintaining 100% sequence identity at the protein level, possessed a sequence identity at the DNA level of ~70%. The aligned DNA sequences for each of the four altered repeats are shown alongside Protirelin the secondary structure notations (Figure 1). The cache of potential codons for each residue was limited by the expression preference of for certain codons as well as the need to prevent RNA secondary structure that could impair translation. Therefore the final construct was a compromise between codon optimization, GCC content and sequence non-identity at the DNA level. RII tetra-tandemer is monodisperse and has an extended conformation in the presence of Ca2+ We have previously shown that the RII-tandemer is fully structured in 10 molar equivalents of Ca2+ but resembles a random coil in the absence of this ion . Similar analyses were applied to the RII tetra-tandemer. In the presence of EDTA, the RII tetra-tandemer appeared to be unstructured with its far-UV CD spectrum displaying a single negative peak at 198?nm (Figure 2A). When the CD spectrum was.