FlhF is a member of the SRP GTPase subfamily of small G proteins. The NG domain of FlhF (NG-FlhF) was predicted to include amino acids 79–366 by multiple sequence alignments and secondary-structure prediction using ClustalX
(Thompson et al.
) and ProteinPredict
(Rost et al.
), respectively. NG-FlhF was expressed and typical yields were about 50 mg per litre of culture (corresponding to 8 g of cells). Protein purification took place according to standard protocols as described above. The protein purity prior to crystallization was >90% as judged from Coomassie-stained SDS–PAGE (not shown).
Initial crystals of NG-FlhF grew in the presence of 5 mM GDP from hanging drops with a reservoir solution containing 10%(w/v) PEG 6000, 4%(v/v) 2-methyl-2,4-pentanediol and 0.1 M Tris pH 7.5. Numerous bipyramidal crystals appeared within a few hours and grew to full size within 2 d (Fig. 1
a, crystal form A). The crystals were not very stable and decomposed after about one week. Only a fraction of them were suitable for X-ray analysis; they diffracted to 3.2 Å resolution at beamline ID29 at the ESRF in Grenoble. When NG-FlhF was crystallized in the presence of 5 mM GMPPNP with a similar reservoir solution consisting of 13%(w/v) PEG 6000, 4%(v/v) 2-methyl-2,4-pentanediol and 0.1 M MOPS pH 7.5, a large number of crystals appeared within 2 d. They exhibited a similar bipyramidal shape (Fig. 1
b, crystal form B) and were unstable; similar to the crystals obtained with GDP, only a few of them showed diffraction. A data set could be collected to 3.2 Å resolution. However, these crystals did not decompose and after three to five weeks a change in crystal morphology was observed (Fig. 1
c). These new crystals (crystal form C) were of homogenous quality and diffracted to a resolution of 3.0 Å. It is important to note that the addition of GDP or GMPPNP was a prerequisite for successful crystallization, as no crystals were obtained in the absence of nucleotide.
Figure 1 Various crystals of B. subtilis FlhF. (a) Bipyramidal crystals grown in the presence of 5 mM GDP (crystal form A, Table 1). (b) Crystals grown in the presence of 5 mM GMPPNP show a similar bipyramidal shape (crystal form B, Table (more ...)
For X-ray data collection, crystals were transferred to a glycerol-containing cryoprotection solution and subsequently flash-cooled as described above. The use of various cryoprotection agents such as ethylene glycol, medium light paraffin oil or sucrose did not improve diffraction. Data collection was performed at beamlines ID29, ID14-1 and ID14-2 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. Three data sets were collected; statistics and crystal data are summarized in Table 1. Notably, crystal forms A
are basically identical in unit-cell parameters and diffraction quality, despite being obtained in the presence of GDP or GMPPNP, respectively. This suggests that the addition of these nucleotides does not influence crystal packing. Crystal form C
belongs to a different space group and the Matthews coefficient (Matthews, 1968
) suggests the presence of eight molecules in the asymmetric unit, while crystal forms A
contain one molecule in the asymmetric unit (Table 1). As the transition from crystal form B
only occurs with the nonhydrolysable GTP analogue GMPPNP, it might be a consequence of slow nucleotide binding or exchange. Analysis of the self-rotation function of the data from crystal form C
shows that the crystal forms A
) and C
are not related (not shown).
Summary of preliminary crystallographic analysis
Our study describes the expression, purification and preliminary crystallographic analysis of B. subtilis FlhF. We expect that the structure will be solvable by molecular replacement using SRP54 or FtsY as a search model. With the structure of FlhF, all three members of the SRP GTPases will be characterized. The structure should provide insights into the regulation of SRP GTPases in general and provide a molecular basis for the role of the third SRP GTPase in flagellar assembly.