1. Volume 24, Issue 7, Pages 1178-1191 (July 2016)
PSCD Domains of Pleuralin-1 from the Diatom Cylindrotheca fusiformis: NMR Structures and Interactions with Other Biosilica-Associated Proteins 
Silvia De Sanctis, Michael Wenzler, Nils Kröger, Wilhelm M. Malloni, Manfred Sumper, Rainer Deutzmann, Patrick Zadravec, Eike Brunner, Werner Kremer, Hans Robert Kalbitzer 
Structure 
Volume 24, Issue 7, Pages (July 2016)
DOI: /j.str
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2. Structure 2016 24, 1178-1191DOI: (10.1016/j.str.2016.04.021)
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3. Figure 1 Pleuralin-1 Protein from C. fusiformis
(A) General structure of pleuralin-1 protein.
(B) Sequence comparison of the five PSCD domains of the pleuralin-1 protein. The sequence identities to the target domain (PSCD4 domain) are highlighted by shaded rectangles while the unaltered positions of the ten cysteine residues are represented by blank boxes. The PSCD4 domain encompasses the residues E372 to P458 that correspond to E16 to P102 in the initial His6PSCD4 construct.
Structure  , DOI: ( /j.str )
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4. Figure 2
Disulfide Bridges and Hydrogen Bonds in the His6PSCD4 Construct
(Top) Fragments contained from limited proteolysis and analyzed by Edman degradation and MS. (Bottom) The sequence of the His6PSCD4 construct made up of 112 amino acids. Gray, His tag; green, six residues of the PSCD3 domain and ten residues belonging to the linker between the PSCD4 and PSCD5 domains; cyan, disulfide bridges determined by limited proteolysis, Edman degradation, and MS; red, hydrogen bonds detected directly by an HNCO experiment (dashed lines) or by analysis of the NOE and hydrogen exchange patterns (solid lines). See also Figure S3.
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5. Figure 3 3D Structure of the PSCD4 Domain of Pleuralin-1 Protein
The structure was calculated as described in Experimental Procedures with the experimental restraints (Table 1) and the substitute restraints (Table S2), and was refined in explicit water. The lowest-energy structure from E16 to P102 is shown. Disulfide bonds are depicted in orange, the secondary structures are green, and the electrostatic surface potentials are red (negative charges) and blue (positive charges).
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6. Figure 4 Internal Mobility in PSCD4
The sample contained 0.8 mM His6PSCD4 in 10 mM sodium phosphate buffer (pH 6.5). Heteronuclear 15N,1H NOEs (gray bars) were measured at a proton frequency of 500 MHz at 298 K and are plotted as function of the sequence position. P, proline residues; 0, amino acids where a reliable NOE could not be determined because of peak overlap or insufficient signal-to-noise ratio in the saturated experiment.
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7. Figure 5
3D Structures of the Five PSCD Domains and the Complete PSCD Region of Pleuralin-1
(A) The 3D structures of the core region of the five PSCD domains. They were calculated assuming that the experimental restraints from PSCD4 can be applied for residues that are strictly conserved in comparison with the sequence of PSCD4. The structures were simulated with CNS using both experimental and substitute restraints. In each case the ten lowest-energy structures are superposed. The numbering of the residues used here corresponds to the complete sequence of pleuralin-1.
(B) Surface representation of the PSCD chain of pleuralin-1. The electrostatic surface potential is indicated with positive (blue) and negative (red) surface charge. Note that information on the structure of the linker regions was not available for the molecular dynamics simulations.
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8. Figure 6 Calcium Binding to His6PSCD4
(A) Plot of the cross-peak combined chemical-shift changes Δδcomb in the 1H,15N HSQC spectra of 0.8 mM His6PSCD4 in 5% D2O and 0.1 mM DSS after addition of 10 mM CaCl2. P, proline residue; 0, cross-peak shift could not be quantified due to low signal-to-noise ratio; ∗, no chemical-shift change observable; M, no chemical shift assigned to those residues. Green and blue lines represent the SD to zero σ0 and 2σ0, respectively.
(B) Surface representation of the PSCD4 domain showing residues with Δδ >2 σ0 in red. The not observable residues including the prolines are depicted in gray. The secondary structure is plotted under the transparent surface using the above-described colors and highlighting the side chains of the charged residues (D34, E75) involved in the two Ca2+ binding sites. An additional Ca2+ binding site was located close to C24 with a chemical-shift change Δδ >2 σ0.
See also Figure S1.
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9. Figure 7 Interaction of His6PSCD4 with α-Frustulin
(A) Plot of the relative cross-peak intensity changes (Io-I)/Io in the 1H,15N HSQC spectra of 0.8 mM His6PSCD in 5% D2O and 0.1 mM DSS after addition of 0.5 mM frustulin in the same buffer. Measurements were performed at 298 K. I0 and I, cross-peak intensity before and after addition of frustulin, respectively. Intensities were corrected for dilution effects. P, proline residue; 0, cross-peak intensity could not be quantified; M, no chemical shift assigned to those residues. Green and blue lines represent the SD to zero σ0 and 2σ0, respectively.
(B) Surface representation of the PSCD4 domain showing residues with (I0-I)/I0 ≤ σ0 and 2σ0 < (I0-I)/I0 in blue and red, respectively. The not observable residues including the prolines are depicted in gray.
See also Figure S3.
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10. Figure 8 Interaction of His6PSCD4 with Native Silaffin-1A
(A) Plot of the combined chemical-shift changes Δδcomb (Schumann et al., 2007) in the 1H,15N HSQC spectra of 0.8 mM His6PSCD in 5% D2O and 0.1 mM DSS after addition of 1.82 mM native silaffin-1A in the same buffer. Measurements were performed at 298 K. P, proline residue; 0, cross-peak chemical shift could not be quantified. Green and blue lines represent the SD to zero σ0 and 2σ0, respectively.
(B) Surface representation of the PSCD4 domain showing residues with Δδcomb ≤ σ0, σ 0 < Δδcomb ≤2σ0, and 2σ0 < Δδcomb in blue, orange, and red, respectively. The not observable residues are depicted in gray.
Structure  , DOI: ( /j.str )
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11. Figure 9
Model of the Mutual Arrangement of Pleuralin, Silaffin-1A, and α-Frustulin in the Overlap Region of Hypotheca and Epitheca
Structure  , DOI: ( /j.str )
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