Supplementary information
Methods
Over-expression and purification of NhaA
Cells of the E. coli strain RK201 transformed with the plasmids pAXH encoding
His-tagged NhaA2 and pIQ (a pACYC184 derivative) encoding LacIQ were used for
over-expression of NhaA. The gene nhaA was deleted from RK20, a property that was
found essential for the purification of NhaA. Protein was produced3 in RK20/pAXH/pIQ
cells after induction (at A600 = 0.6) with 0.5 mM isopropyl-β-D-thiogalactopyranoside
for 2 h at 37 ºC. Cells were harvested and high pressure membranes3 were prepared and
frozen (10 mg membrane protein/ml) in liquid nitrogen and stored at –80 ºC. Protein
was extracted from the membranes at 4 ºC for 1 h using a solution containing 1 % ndodecyl-β-D-maltopyranoside, 100 mM MOPS/KOH (pH 7), 28% glycerol, 3 mM
Tris/Cl, 83 mM sucrose and 47 mM choline chloride. The protein was bound to a Ni2+NTA affinity column and purified as described3. On the column, the detergent was
exchanged to 0.03 % n-dodecyl-α-D-maltopyranoside in 20 mM Bis-Tris (pH 6.5).
Elution was conducted at acidic pH (25 mM Na+/citrate, 5 mM MgCl2, 100 mM KCl,
pH 4). The protein was concentrated (Centriprep YM-50) to 12 mg/ml and frozen in
liquid nitrogen in the above solution supplemented with 20 % sucrose and stored at –80
ºC. For protein labelled with seleno-L-methionine, E. coli B834DE39 (Novagen, WI,
USA) transformed with pAXH and pIQ were grown in minimal medium (L-methionine
50 µg/ml replaced by seleno-L-methionine) and the protein was processed as above.
Crystallisation
Crystals were grown by hanging-drop vapour diffusion at 6 ºC by mixing equal
volumes of protein (4-6 mg/ml) and reservoir solutions. NhaA crystals were grown over
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a reservoir solution of 28-34% polyethylene glycol 400, 200-450 mM MgCl2, 100 mM
KCl, 25 mM Na+-citrate (pH 4), 1% n-octyl-β-D-glucopyranoside and 0.5 % ethanol.
Crystals appeared in 4 days and reached a maximal size of 0.2 x 0.3 x 0.3 mm in 15
days. The crystals were gently removed from the drop and flash frozen with nitrogen
(100 K).
Data collection and structure determination
Diffraction data were collected at the ESRF beam lines ID29 (data set SeMet
SAD) and ID23 (data set Native 1) and at the SLS beam line PX06 (data set Native 2)
with charge-coupled device detectors and cryo-cooling (100 K). Data were processed
and scaled using the programs HKL4, XDS5 and the CCP4 program suites6. The crystals
belong to an orthorombic space group P212121. Two molecules NhaA are present in the
asymmetric unit. The structure was solved by SAD technique using 20 selenium atoms.
Initial positions were found using Solve7 and were refined with SHARP8. Phases were
calculated to 4.3 Å (17 - 4.3 Å) using SHARP with a figure of merit of 0.42 for acentric
reflections and 0.13 for centric reflections, a phasing power (anomalous) of 2.4 and
Rcullis (anomalous) of 0.52 for acentric reflections. The SAD phases were applied to the
native data set (native1) and gradually extended to 3.8 Å using the program DM 9 with
solvent flattening, histogram matching and averaging options. The correlation between
the electron density areas related by non-crystallographic symmetry was 78.8 % after
density modification. An initial model was built using the program O10 and refinement
was carried out using CNS11 with tight two-fold NCS restraints. For final refinement of
the model the second native data set at 3.45 Å was used.
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Fig. 1 and 3b were prepared using the program O, Fig. 2, 4, S1 and S2 were made with
MOLSCRIPT12 and rendered with RASTER3D13. Fig. 3a was prepared using Pymol14
and Fig. 3b-d with the program GRASP15
References Supplementary Information
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