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   Table I

   Data collection and phasing statistics
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   Table II

   Refinement statistics

 §5§ Data Collection and Structure Determination of CoA-bound bPanK §5§

   Complete diffraction data from a single crystal of the CoA-bound bPanK
   were also measured at beamline X12C at the National Synchrotron Light
   Source. The programs DENZO and SCALEPACK ([66]14) were used to
   integrate and scale the data set. The crystals of the CoA-bound enzyme
   belong to space group P1 with unit cell dimensions ofa = 62.0 Ã…, b =
   71.2 Å,c = 87.7 Å, α = 102.4°, β = 89.5°, and γ = 93.2°. The data
   collection statistics are summarized in Table [67]I.

   The structure of the CoA-bound bPanK was determined by the molecular
   replacement method using one subunit of the AMPPNP-bound enzyme as a
   search model. The cross-rotation and translation functions and
   Patterson correlation refinement were calculated using the program
   XPLOR ([68]19). Using the data between 15.0 and 4.0 Ã…, the rotation
   function, followed by Patterson correlation refinement, gave four
   outstanding solutions that correspond to four subunits of bPanK.
   Because any point can be taken as an origin in space group P1, rotation
   of the initial search model according to one of the four rotation
   function solutions determined the orientation and position of the first
   subunit. With fixing this orientation and position of the first
   subunit, the orientations of the other three subunits were determined
   by applying the corresponding noncrystallographic symmetry operations
   to the first subunit. The positions of the other three subunits were
   then determined by finding the relative x, y, andz translations of each
   of the three subunits with respect to the first subunit. The model
   including all four subunits was subjected to rigid body refinement at a
   resolution between 6. 0 and 4. 0 Ã… in the program XPLOR ([69]19). The
   high resolution limit, which restricted the maximum shift of structure
   in the refinement, was set to 4. 0 Ã… resolution to provide a possible
   large shift of the structure up to 4.0 Ã…. The low resolution limit was
   set to 6.0 Ã… resolution below which the intensities of reflections were
   severely affected by the diffraction of solvent molecules in the
   crystal. The Rfactor at this stage was high (51.0%), suggesting that
   the conformation of the CoA-bound structure was different from that of
   the search model, the AMPPNP-bound structure. To consider the solvent
   contribution to the intensities of reflections at low resolution, the
   structure factor amplitudes of solvent molecules in the crystal were
   calculated using the bulk solvent correction routine in XPLOR ([70]19),
   and thus the low resolution limit could be extended to 50.0 Ã…
   resolution. Crystallographic refinement including simulated annealing,
   conjugate gradient minimization, and individual B-factor refinement was
   then performed at the resolution between 50.0 and 2.5 Ã…. After the
   first round of refinement, the quality of the structure was
   dramatically improved with a working R factor of 27.4% and a freeR
   factor of 30.1%. The F [o] −F [c] difference map showed continuous
   density for CoA, verifying the correct molecular replacement solution.
   Several iterations of model building in the program O ([71]17) and the
   refinement in the program XPLOR ([72]19) further dropped both R
   factors. The final refinement statistics are shown in Table [73]II.
   Because of the slight difference between four subunits,
   noncrystallographic symmetry restraints were applied to only 89% of
   total residues of each subunit. In the final model, residues 1–5 in
   subunit 1; residues 1–7 and 211–212 in subunit 2; residues 1–5 and
   210–213 in subunit 3; and residues 1–7, 83–85, and 212–214 in subunit 4
   are excluded because they are disordered in the crystal.
   [74]Previous Section[75]Next Section

 §2§ RESULTS §2§

 §5§ Monomer Structure §5§

   In the structures of the AMPPNP-bound and the CoA-bound bPanKs, four
   identical subunits are found in an asymmetric unit. When the four
   subunits occupying an asymmetric unit of each structure are compared,
   the average RMSD of Cα atoms for the AMPPNP-bound and the CoA-bound
   enzymes are 0.46 and 0.34 Ã…, respectively. These values are consistent
   with estimated errors in their atomic coordinates. A subunit of bPanK
   adopts a mononucleotide-binding fold ([76]20): a seven-stranded β-sheet
   (strands, 2, 3, and 8–11) is flanked by α-helices (D and E on one side
   and G and J on the other side) (Fig.[77]1). An intervening loop between
   strand 2 and helix E, known as the P-loop, contains most of the
   residues that interact with phosphate oxygens of the AMPPNP. There are
   four small antiparallel β-strands (strands 4–7) that are not part of
   the main β-sheet (Fig. [78]1 a). Residues from helices H and I
   including their intervening loop are involved in binding CoA (Fig.[79]1
   a). The N-terminal region including strand 1, helices A and D, and a
   loop between helices A and B forms the major part of the dimer
   interface (Fig. [80]1 a).
   [81]Figure 1
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   Figure 1

   A subunit structure of the AMPPNP-bound bPanK. a, schematic
   representation. Helices are shown in red, strands are in yellow, and
   other secondary structural elements are in green. The N-terminal region
   involved in dimerization is shown in blue. AMPPNP (ball-and-stick
   representation) is shown in yellow. Figs.[85]1 a, [86]2, [87]3 b, [88]4
   b, and [89]5 were prepared using InsightII (Molecular Simulations). b,
   topology representation. Cylinders and arrows represent α-helices and
   β-strands, respectively. c, the sequence alignment of bPanK with
   prokaryotic PanKs fromStreptococcus pyogenes, Enterococcus
   faecalis,Pasturella multocida, Hemeophilus influlenzae,Actinobacillus
   actinomycetemocomitans, Salmonella typhi, Klebsiella pneumoniae,
   Yersinia pestis, Vibrio cholerae, Mycobacterium tuberculosis,
   Corynebacterium diptheriae, andStreptomyces coelicolor. The sequence
   numbering and secondary structure assignment is according to the bPanK
   sequence. Amino acid residues identical in 12 of 13 sequences are shown
   inred. Residues implicated in catalysis are marked withasterisks, and
   Lys^101 involved in binding of ATP and CoA is marked with a triangle.

   The Dali server ([90]21) was used to search for known proteins
   structurally similar to bPanK. Applied to one subunit of bPanK, the