Locali crysta ization an als using nd orienta molecula ation of h ar ...

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Acta Crystallographica Section D research papers
3.1. Preparation of hexa-sodium �-metatungstate-DR6 derivative crystals
We have successfully obtained isomorphous DR6 derivative crystals by soaking them in stabilizing
solution supplemented with 5 mM of HMT. This HA-cluster is composed of twelve tungsten atoms
showing a structure similar to the more commonly used TPT (Figure 1). The phosphate core of the
latter is replaced by four water molecules in HMT resulting in a higher overall charge of -6 (Fig. 1A,
B). Both clusters are characterized by an internal tetrahedral symmetry but differ in the W-O-W bond
angles as a result of a differently isomerized WO4 - - substructure. HMT is soluble in acidic, in neutral
and in slightly basic aqueous solutions with high or low ionic stren gth. In contrast, other HA-cluster
compounds tested (TaB and TPT) showed lower solubility under the tested conditions. As low
solubility is often a limiting factor for the practical use of derivatisation agents, this observation
favors the broad application of HMT (Hastings & Howarth, 1992).
Tungsten displays strong anomalous diffraction of 5.6 electrons at the copper K�-wavelength and a
strong white line at its L-III absorption edge (1.2147 Å). Hence successful derivatisation of DR6
crystals with HMT could be monitored by a significantly increased anomalous correlation coefficient
(CCanom) of the in-house data. The unit cell constants of the native and the derivative crystals (Table 1,
SIRAS) show only slight differences and the corresponding datasets indicated a sufficient degree of
isomorphism (Supplementary information). Three additional datasets were collected from the same
derivative crystal at the tungsten L-III absorption edge to perform a three wavelength MAD
experiment (Table 1, MAD).
3.2. Orientation and localization of HMT by molecular replacement
To orient the HA-cluster and thus to localize the individual tungsten atom positions of HMT, we
applied molecular replacement (MR) in MOLREP (Vagin & Teplyakov, 1997). The coordinates of the
Cambridge structural database (CSD) entry ABOCIE (Allen, 2002) were directly used as the search
model. MOLREP was run with default settings via the CCP4i-interface, disabling the packing and the
scoring system. Instead of structure factor amplitudes either the isomorphous differences of the inhouse
datasets (Table 1, SIRAS) or the anomalous differences of the MAD-peak dataset (Table 1,
peak) were used. To test the performance of this procedure at different resolutions, the respective
datasets were cut in 0.5 Å steps and the MR-calculations were repeated. Correct solutions were well
separated from noise by significantly higher score and contrast (TFcnt) values in MOLREP (Table 2)
and were found for isomorphous as well as anomalous differences down to resolutions as low as 4.4 Å
and 4.9 Å, respectively. In addition, we could also identify “partial solutions” represented by
intermediate scoring values. In this case the correct center of mass but an incorrect orientation of the
HA-cluster was identified, resulting in incorrect placement of the individual tungsten atoms. Such
“partial solutions” were obtained down to 5.4 Å resolution using anomalous differences.
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