Data collection statistics.

<p>Numbers in parentheses belong to the outer shell.</p>†<p>R_merge  =  ∑_hkl∑_i |I_i(hkl) - |/∑_hkl∑_i I_i(hkl), where I_i(hkl) is the observed intensity for a reflection and is the average intensity obtained from multiple observations of symmetry-related reflections.</p>‡<p>R_meas  =  ∑_hkl [N/(N-1)]^1/2 ∑_i |I_i(hkl) - |/∑_hkl∑_i I_i(hkl), where I_i(hkl) is the observed intensity for a reflection, is the average intensity obtained from multiple observations of symmetry-related reflections and N is the number of observations of intensity I(hkl).</p><p>Data collection statistics.</p

Surface representation of GyrB43 demonstrating the deeply buried nucleotide (stick model).

<p>View along the narrow tunnel leading to nucleotide. (a) GyrB43⋅AMPPNP complex, (b) GyrB43⋅ADP⋅P_i complex. The insets show close-ups of the nucleotide sites with the ATP lid loop (residues 99–120) and the adjacent subunit of the dimer in surface representation (same colour codes as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107289#pone-0107289-g002" target="_blank">Fig. 2</a>). Note, that upon ATP hydrolysis glutamine 335 cannot escape "downwards" to relieve a clash with the P_i moiety due to the presence of the ATP lid. Instead the entire transducer domain moves to the side.</p

Pair-wise fit of GyrB43 domains after superposition of the ATPase domains (regular) or transducer domains (italics).

<p>The values give the rmsd (Å) of the corresponding Cα positions. When superimposing the ATPase domains dimer, the pairwise rmsd range from 0.46 Å to 0.93 Å.</p><p>Pair-wise fit of GyrB43 domains after superposition of the ATPase domains (regular) or transducer domains (italics).</p

Domain architecture of <i>E. coli</i> DNA gyrase and model of DNA gyrase mechanism.

<p>(a) Domain architecture of <i>E. coli</i> DNA gyrase. GyrB is composed of an ATPase (yellow), a transducer (orange) and a toprim (red) domain. GyrA is composed of a winged-helix (WHD, blue), a tower (blue), a coiled-coil (light purple) and a β-pinwheel (β-PW, purple) domain. (<i>b</i>) A double-stranded DNA segment (G-segment) is captured at the DNA-gate, the N-gate is open to allow access of the T-segment. (<i>c</i>) Upon ATP binding, the ATPase domains dimerize and the T-segment gets trapped. (<i>d</i>) This is followed by ATP hydrolysis, G-segment cleavage, DNA-gate opening and T-segment translocation. The domain colored in light-grey represents the transducer domain in the preceding orientation. The mechanistic details of this step are not clear, in particular whether prior to P_i release the enzyme conformation is changed and how ATP hydrolysis and DNA passage are coordinated. For clarity, the coiled-coil and β-pinwheel domains of GyrA are omitted in (b-d). Adopted from references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107289#pone.0107289-Sissi1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107289#pone.0107289-Gubaev1" target="_blank">[5]</a>.</p

Refinement statistics.

<p>Numbers in parentheses refer to the outer shell.</p><p>* <i>R_work</i>  =  ∑_hkl|| F_obs| - |F_calc||/∑_hkl|F_obs|</p><p>** R_free is the R value calculated for 5% of the data set that was not included in the refinement.</p><p>*** Molprobity.</p><p>Refinement statistics.</p

GyrB43 crystal packing of the P2₁2₁2 (left, GyrB43⋅AMPPNP complex) and the C222₁ (right, GyrB43⋅ADP⋅P_i complex) form.

<p>(a) The molecular packing is shown within a slab perpendicular to b and centered at y = 1/4. In both forms, dimers are oriented with their molecular dyads parallel to b (viewing direction). In the P2₁2₁2 form (left) the molecular dyad is local (green elliptical symbol), in the C222₁ form (right) the molecular dyad is crystallographic (black symbol). In each case, neighbouring dimers are related by horizontal 2-fold screw-axes. Black spheres represent the position of residue D377 at the end of the transducer domain (also depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107289#pone-0107289-g004" target="_blank">Fig. 4b</a>). (b) Details of the major crystal contact formed in both packings between the transducer domain and the ATPase domain of a symmetry related dimer. (c) Representation of the symmetry elements, same view as in panel (a). Unit cells are indicated by solid line. With respect to the arrangement of symmetry elements, the dashed rectangle of the scheme at the right is equivalent to the unit cell of the left scheme. Local symmetry elements are indicated in green.</p

Structure comparison of (a) GyrB43⋅ADP⋅P_i (magenta) and (b) GyrB43⋅ADP⋅BeF₃ (violet) with GyrB43⋅AMPPNP (shown as a dimer with yellow/grey colour with molecular dyad in black).

<p>The structures are superimposed on their ATPase domain (residues 20–220). The rotation axes for the domain reorientation of the transducer domain with respect to the ATPase domain are indicated. These form an angle of 21.5° and 14.5° with the molecular dyad for GyrB⋅ADP⋅P_i and GyrB⋅ADP⋅BeF₃, respectively. The insets show the QTK loop of the transducer domain that is rotated relative to the ATPase domain in response to the nucleotide state (dashed lines); Q: Q335, K: K337. (c) Stereoview of the overlay of the three aforementioned structures.</p

Quantification of nucleotide induced structural changes within dimeric GyrB43.

<p>(a) Inter-subunit distance changes between symmetry related residues of selected transducer domain Cα-atoms. The changes have been calculated relative to the AMPPNP complex structure, green bars indicate an increase in distance, red bar indicate a decrease. (b) Cartoon representation of dimeric GyrB43 (ADP⋅P_i state) with the residues used for the calculations in (a) indicated. View along the symmetry axis from the C-terminal side.</p

Refined mechanistic scheme of DNA gyrase activity, based on reference [2], representation as in <b>Fig. 1.</b>

<p>The N-gate of the GyrA₂GyrB₂ heterotetramer with bound G-segment at the central DNA-gate (top) closes upon ATP binding thereby trapping a T-segment in the upper chamber (step a). Hydrolysis of the two ATP molecules causes a 12° rotation of the respective transducer domains relative to the ATPase domain (step b). We propose that this conformational change is coupled to DNA gate opening and T-segment translocation. Subsequent P_i release would be coordinated with G-segment re-ligation and DNA-gate closure (step c). Finally, ADP release results in dissociation of the ATPase domains and a reset of the enzyme (step d).</p

Conformational states of GyrB43 along the ATP hydrolysis reaction path.

<p>The various states have been trapped by co-crystallization with the appropriate nucleotides. The dimeric structures are shown in cartoon representation with semi-transparent molecular surface overlaid. Subunits are distinguished by a slight variation in colour hue. View perpendicular to the molecular dyad. (a) AMPPNP complex (PDB entry 1EI1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107289#pone.0107289-Brino1" target="_blank">[13]</a>), (b) ADP⋅P_i complex, (c) ADP⋅BeF₃ complex and (d) ADP complex. Note the distinct opening angles defined by the two transducer domains with the AMPPNP and ADP complexes in a "closed" conformation, the ADP⋅BeF₃ complex in a "semi-open" and the ADP⋅P_i complex in "open" conformation.</p