Image processing

Images of crystals were taken at magnification of 70,000x with the specimen grid tilted between 0 and +45^o within the microscope. A single image was taken per crystal tube. Micrographs of crystals that diffracted to at least 3 orders and lacked excessive astigmatism and drift were chosen for digitization. Images of the flattened tubes showed two easily distinguishable crystalline lattices rotated by 12°-15° which originate from the two sides of the flattened tube, Images were processed using the MRC suite of programmes. These programmes reduced lateral lattice distortion in the crystal, and increased the signal to noise ratio by the process of lattice unbending and quasioptical filtering (Crowther, 1996). In most cases, both lattices observed from the images of flattened tubes could be processed separately and used for the 3D reconstruction. The lattices possessed p22121 symmetry with unit cell dimensions a = 81°, b = 108°, γ=90°. Selected images of tilted specimen with their corresponding computed Fourier transforms generated after processing (2-3 rounds of unbending) are shown in Figure 31.

Figure 31 Fourier transforms of typical images recorded at tilt angles (A) 0°, (B) 20°, (C) 30° (D) 45º. Circles denote 12,10, 8, 6 and 4 Å resolutions, respectively

The merged data set was evaluated with the program LATLINEK (Agard, 1983), which generates lattice lines (Figure 26) by applying a weighted least-squares procedure to the phases and amplitudes. Due to the limited amount of points for comparison, some-out-of scale amplitudes were observed. These out-of-scale amplitudes were manually modified. Curves were fitted to the plotted data and sampled at ΔZ =(1/150) Å-1.

A B

C D

Figure 32 Plots of amplitudes (lower panels) and phases (upper panels) along the z* axis for three selected reflections. The fitted lattice lines were produced by weighted least squares fitting and the resulting errors are shown.

Table 2 Crystallographic data summary

Plane group p22121

Number of images* 74 Resolution in plane^a 7Å Resolution vertical^a 14Å-15Å Total no. of observations^b 7475 No of unique observations 1494 Overall weighted R-factor (%)^b 0.297 Overall weighted phase error^b 17.8°

*Distribution of tilt angles (0°-15, 20°-14, 30°-17, 45°-28)

a As calculated from a point-spread function of the experimental data.

b From program LATLINEK.

3.7 3D map of MjNhaP1

The phases and amplitude obtained from the fit lattice lines were combined to generate a 3D map of MjNhaP1. Four different maps were calculated applying B-factors of -350 Ų, -400 Å^2,, -450 Ų and -500 Ų respectively. The maps calculated with B-factors 400 Ų and 450 Ų showed fully resolved helical densities and these two maps were very similar. The map calculated with -500 Ų had the lowest apparent signal to noise ratio.

The projection map (Vinothkumar et al., 2005) had shown MjNhaP1 as a dimer with dimenions of ~51Å x 84Å. The 3D map of MjNhaP1 also shows a tightly packed dimer with an oval shape ( Figure 33). In the 3D map, the dimensions of MjNhaP1 in the membrane plane are ~95 Å for the long and ~50 Å for the short axis. In the side-view, the MjNhaP1 has a thickness of ~45 Å. As mentioned in table 2 the in-plane resolution of the map is 7Å and the resolution in the vertical direction is 14Å. At this resolution, elongated helical densities could be clearly observed. These helical densities formed two distinct regions as shown by the delineated boundary of one monomer ( Figure 33). The two distinct regions identified are:

a) The region in the central part of the dimer composed of several tilted helices forming an interface between both monomers. Four helices in the centre could be

monomer two or three helical densities appear to be present. These two or three helices are in close contact with the second monomer.

Figure 33 3D electron density map of MjNhaP1. The map is contoured at 1σ (blue) and 2σ (purple). (A) Top view, one monomer is highlighted. The outer bundle and dimer interface are highlighted in green and yellow respectively. (B) The side view shows the NhaP1 map along the membrane plane.

b) In the other region, a group of six tightly packed helices form an outer bundle. This bundle contains three helices in the centre of the monomer and three helices on the outermost periphery. The three helices in the centre are almost perpendicular to the membrane plane, clearly separated from each other and extend from the top to bottom of the map. The densities for the other three peripheral helices are more connected to each other. They tilt at an angle of 5°-10° from the membrane normal.

There is a stretch of density in the centre connecting the three peripheral helices to three central one.

3.8 Sequence alignment

To investigate, whether the distinguishing structural features of MjNhaP1 could be a common feature of other archaeal, bacterial and eukaryotic Na⁺/H⁺ exchangers/antiporter (2.13), alignment of several amino acid sequences was carried out. The alignment indicated a different number of hydrophobic segments in different families. The NhaA family from E.coli, S.enterica and V.aginolytiicus contain 12 hydrophobic segments. The archaeal MjNhaP1 families show 13 hydrophobic segments. Finally, the NHE and Nhx families from eukaryotes show 14 hydrophobic segments with the exceptions of Nhx1 from A. thaliana and the human Nhe6a which have 13 and 15 hydrophobic segments, respectively. The alignment revealed that the amino acid sequence for the β-sheet of the X-ray structure of NhaA (Hunte et al., 2005) is present only in the two other bacterial species. This region is missing in the eukaryotic and archaeal counterparts.

The corresponding hydrophobic segments from the same family show high sequence conservation. Sequence conservation is also observed for the intermediate hydrophilic stretches of the same family especially for the prokaryotic proteins.

Sequence motifs common to all families have been found in segments corresponding to transmembrane helices IV and V of the NhaA X-ray structure. Helix IV in NhaA is unwound in the sequence AIPAATDI located in the middle of the membrane (Hunte et al., 2005). A similar motif appears to exist in all other antiporters as shown in the alignment. The other unwound helix XI in NhaA disrupts a GIG motif. However, such a motif is not present in any other antiporter. Instead, a conserved GPRGVVP and GGLRG motif is present in the NhaP and NHE family respectively, with Nhx1 from S.cerevesiae and A. thaliana as exceptions. Helix V also shows high conservation in all the selected families in the alignment due to the presence of the common motif AIDDLG, FNDPLG and NDA in the NhaA, NhaP and NHE family, respectively.

Figure 34 Alignment of Na⁺/H⁺exchangers. Nomenclature as for putative helices in NhaP1. NhaA helices in Roman numerals in bracket.

The archaeal antiporters share conserved sequences with both their eukaryotic and prokaryotic counterparts. The amino acids in hydrophobic segments corresponding to helix IX of NhaA show conservation in the archaeal and eukaryotic antiporters. On the other hand, helix X of NhaA contains the motif KPLG which represented by the RPLG motif in the NhaP family.