1
Supplementary Information
Supplementary Data
ParB proteins from different partition systems show little sequence homology and bind distinct types of
partition-sites. A detailed molecular understanding of partition complex formation has been hampered by
the dearth of structural information available for ParB proteins, which is perhaps due to their inherent
flexibility. Structures have only been available for short regions, including the HTH-domains, of the
Thermus thermophilus Spo0J and RP4 KorB, which both recognize single inverted repeat partition-sites,
unlike P1 ParB which recognizes a complex multi-Box partition site. Indeed, comparisons of the three
proteins show that only their HTH units (1-3 in P1 ParB) have structural similarity. Whether ParBlike proteins that recognize only inverted partition-site repeats utilize a bridging DNA-binding
mechanism like P1 ParB is unknown as the structures of the DNA-binding/dimerization regions of these
proteins have not been solved together.
Supplementary Methods
Full-length E. coli P1 ParB, with or without DNA, did not crystallize under any condition tested and was
proteolytically labile. Thus, P1 ParB(142-333), which contains all the determinants required for DNA
binding, was produced with an N-terminal Hexa-histag, expressed 1in BL21(DE3)pLysS and purified
using two column chromatography steps: Ni-NTA and DE53. Wild type ParB(142-333) was
concentrated to 40 mg/mL and dialyzed into 30 mM Tris pH 7.5, 150 mM NaCl and 5% glycerol for
crystallization. The same buffer was used for the selenomethionine substituted protein except 1 mM ME was added. Dehydration of crystal form I was carried out by moving them to a drop of saturated
Citrate, followed by slow removal of the Citrate solution, which resulted in a shrinking of the c cell edge
to 132.3 Å and improvement of diffraction from 10.0 Å to better than 3.0 Å.
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Supplementary Figures
Figure S1. Overall ParB structure and secondary structure assignment. Shown above the ParB ribbon
diagram is the P1 ParB sequence. In the ribbon diagram, one ParB subunit is grey the other is colourcoded for secondary structure, with -strand, -helices and coil region coloured red, green and yellow,
respectively. The sequences encompassing the secondary structural elements are indicated by red, green
or yellow regions under the ParB sequence, and are labelled.
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Figure S2. The Dimer-domain contains DRS residues critical for centromere-site
binding. Shown in this figure are sequence alignments of ParB proteins from par
systems E. coli P1, Salmonella typhimurium pSLT, Salmonella flexineri pWR501,
Yersinia pestis pMT1 and E. coli P7 that are most related to E. coli P1. Shown to the
right of the protein sequence alignment are the corresponding B-Box sequences from
the parS centromere-like sites for these systems. Conserved residues in the Dimerdomain regions that are identical are red, highly conserved are coloured blue and green.
The B-Box interacting residues that are shown in the ParB-DNA structures to make
base specific contacts to the B-Box are Lys287, Asp288, Arg298 and Arg306. The
“discriminator recognition sequences” (DRS), which include P1 ParB residues K287
and D288, are indicated.
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Figure S3. Model of the dimerized Dimer-domain-double B-Box complex. To generate
the complex, the DNA-bound subunit, with DNA bound, was superimposed onto the
non-DNA bound form and both subunits, with DNA bound, were output. One Dimerdomain subunit is coloured green and the other, red. The DNA is shown as sticks, with
the bases coloured according to atom type with carbon, oxygen, nitrogen and
phosphorous coloured yellow, red, blue and purple, respectively. The 5´ to 3´ direction
of the DNA is indicated in the top figure. Below is a side view of the model. This
figure shows that the two B-Boxes can be simultaneously bound by ParB in the correct
direct repeat arrangement found in parS.
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Supplementary Table 1
SelenoParB(142-333)-25-mer parS-small MAD analysis
Wavelength ()
0.9797
1.0200
0.9796
Resolution (Å)
95.35-3.15
95.35-3.15
95.35-3.15
overall Rsym(%)a
10.8 (31.1)b
9.0 (24.2)
10.9 (34.5)
overall I/(I)
6.7 (2.3)
7.0 (2.9)
6.9 (2.1)
#Total Reflections
106030
101350
105194
#Unique Reflections
16482
16475
16440
Overall Figure of Meritc
0.520
Crystal form
I
II
Resolution (Å)
65.94 - 2.98
75.5 – 3.90
overall Rsym(%)a
9.0 (27.2)
8.2 (41.5)
overall I/(I)
7.7 (2.5)
5.6 (1.8)
#Total Reflections
56031
19307
#Unique Reflections
18057
7020
% complete
96.9 (91.9)
98.2 (97.8)
Refinement Statistics
Resolution (Å)
65.94 – 2.98
Rwork/Rfree(%)d
24.7/29.6
Rmsd
Bond angles (º)
1.62
Bond lengths (Å)
0.010
B-values (Å2)
Average B-values (Å2)
2.7
Ramachandran analysis
Most favored (%/#)
86.5/283
Additional allowed (%/#)
12.8/42
6
Generously allowed (%/#)
0.6/2
Disallowed (%/#)
0.0/0
aR
sym
b
= |Ihkl-Ihkl(j)|/ Ihkl, where Ihkl(j) is observed intensity and Ihkl is the final average value of
values in parentheses are for the highest resolution shell.
cFigure
of Merit = <|P()ei/P()|>, where  is the phase and P() is the phase probability distribution.
= ||Fobs| - |Fcalc||/|Fobs| and Rfree = ||Fobs| - |Fcalc||/|Fobs|; where all reflections belong to a test set
of 5% randomly selected data.
dR
work