Deane et al. 10.1073/pnas.0602689103. |
Supporting Figure 5
Supporting Table 2
Supporting Table 3
Supporting Figure 6
Supporting Methods
Supporting Movie 1
Supporting Figure 7
Supporting Movie 2
Supporting Figure 8
Supporting Figure 9
Fig. 5. Schematic diagram of the type III secretion system (T3SS) apparatus. The gross morphology of the secretion apparatus consists of a cytoplasmic bulb, a transmembrane domain (composed of an inner bacterial membrane ring, periplasmic region, and outer bacterial membrane ring), and an extracellular needle. The pore complex is inserted into the host-cell membrane upon activation. The approximate positions of some component proteins from the Shigella T3SS are indicated.
Fig. 6. The "head" structure of MxiH is conserved between the two crystallographically distinct molecules. The head region (residues 26–57) of MxiH was aligned for molecules A (red) and B (blue). Views rotated by 90° about the long axis of the molecule are shown.
Fig. 7. Relative size of secreted protein and assembled needle channel. (A) End-on view of the C-terminal helix of MxiH (residues 60–80) shown as a surface (green) positioned in the central channel of the A-needle assembly (blue). (B) Side view of A- needle colored as in A with the front subunits of the needle removed. Note that A and B are not at the same scale.
Fig. 8. Sequence alignment of needle subunit proteins from Yersinia and Shigella sp. Residues conserved between Yersinia YscF and Shigella MxiH are shown in red.
Fig. 9. Mapping of residues conserved among T3SS needle subunits onto the surface of the assembled needle. Surface representation of the side view of the needle fitted to the EM map using the A molecule. Conserved residues (21) in the head domain (P44, L46, L47, A48, Q51, L54, and Y57, black) and in the tail domain (N62, K69, K72, and D73, gray) are shown. Each MxiH monomer is colored differently, starting from red and circling the needle to purple.
Movie 1. Rotating view of the atomic model of MxiH docked into the EM density of the Shigella T3SS needle. Side view of the needle assembled using molecule A of MxiH (ribbon) with the modeled N-terminal helix (cylinder). Each MxiH monomer is colored differently, starting from red and circling the needle to purple. EM density is shown as a blue mesh.
Movie 2. Predicted motions of MxiH molecule A. The eigenvector, representing 50% of the motion of the monomer, is represented by a porcupine plot, where the length of the line correlates with the amplitude of the motion.
Table 2. Data collection statistics for SeMet crystal 2
SeMet xtal 2 | |
X-ray source | ID29 |
Detector | ADSC CCD |
Space group | C 2 |
Unit-cell parameters | a = 181.7 Å, b = 28.0 Å, c = 27.7 Å,α = γ = 90.0° β = 96.7° |
Wavelength, Å | 0.9793 (peak) |
Resolution limits, Å | 90.2–2.0 (2.1–2.0) |
Completeness, % | 99.6 (99.6) |
Reflections measured | 94,401 (13,849) |
Unique reflections | 9,670 |
Multiplicity | 9.8 (10.1) |
R merge* | 0.093 (0.223) |
I/σ(I) | 3.7 (2.9) |
Ranom† | 0.030 (0.058) |
Data in parentheses are for the highest-resolution shell.
*Rmerge = 100 × Σh(Σi|<I(h)> – I(h)i|/ΣiI(h)i), where I(h)i is the ith observation of reflection h, and <I(h)> is the mean intensity of all observations of h.
†
Ranom = 100 × Σh|<I+> – <I–>|/Σh(<I+> + <I–>), where <I+> and <I–> are the mean intensities of the Bijvoet pairs for observation h.Table 3. Multiple isomorphous replacement (MIRAS) structure determination statistics
Native | SeMet (xtal1) | SeMet (xtal2) | UO2(AcO)2 | ||
Peak | Remote | ||||
Resolution, Å | 28–2.1 | 28–1.9 | 28–2.3 | 28–2.0 | 28–3.2 |
Wavelength, Å | 0.9340 | 0.9794 | 0.9757 | 0.9793 | 0.9330 |
Isomorphous phasing power | |||||
Acentrics: 28–3.2 Å | 0.283 | 0.134 | 0.320 | 0.890 | |
Centrics: 28–3.2 Å | 0.229 | 0.122 | 0.27 | 0.87 | |
Anomalous phasing power | |||||
Acentrics: 28–3.2 Å | 0.570 | 0.196 | 1.16 | 0.160 | |
Acentrics FOM | |||||
28–9.2 Å | 0.819 | ||||
28–3.2 Å | 0.515 | ||||
Centrics FOM | |||||
28–9.2 Å | 0.616 | ||||
28–3.2 Å | 0.361 | ||||
Supporting Methods
Additional Methods and Crystallographic Software References. Phase determination of the MxiH crystal structure. Phasing was initially attempted with the native and SeMet data sets (Table 2 and ref. 1) and the Se site derived from the anomalous difference Pattersons. These maps showed the correct packing at low resolution but did not allow hand discrimination, nor were they interpretable. A number of native MxiH crystals were then soaked with eight different heavy-atom compounds and data collected from all of them on the fixed wavelength beamline ID14-2 as a preliminary screening test for binding. The uranyl soak was the only one showing clear signal for one site in the isomorphous difference Patterson. A SHARP heavy-atom refinement of this U site against native, uranyl, and SeMet derivative data (with no Se site declared) had reasonable U isomorphous difference signal, allowing location of the Se site on the same origin as the U site: the Se site appeared as a 10 s peak in the SeMet pk2 anomalous log-likelihood gradient maps. The data from SeMet crystal 1 were then added to run the final multiple isomorphous replacement (MIRAS) SHARP job that gave the phases reported here (Table 3), and allowed initial model building.
Superposition of LcrV onto MxiH.
Residues 45–75 of the MxiH A molecule were superposed onto residues 287–317 of the crystal structure of LcrV (2) by using CCP4-lsqkab (3). The same residue ranges were used to superpose LcrV onto each of the five monomers of MxiH at the tip of the needle assembly.Dynamics calculations
. Molecule A of MxiH was submitted to the Dynamite server (4, 5) for prediction of protein motions.References for crystallographic software programs
. Phasing and solvent-flattening were carried out by using SHARP (6), CCP4-DM (7), and SOLOMON (8). The structure was built and refined by using Xfit (9), Buster-TNT (10), and ARP/wARP (11). Calculation of correlation coefficients for the model in EM density was carried out by using MAPMAN (12). All other programs are part of the CCP4 suite (3).Additional Methods. Structural similarities of MxiH to flagellin, EspA, and YscE.
Flagellin is an ≈50-kDa protein that folds into three structurally distinct domains. The D0 portion consists of two helices (one from each terminus of the molecule) that form the central core of the flagellar assembly as visualized in a high-resolution EM reconstruction (13). EspA forms a flagellin-like extension to the hook-analogous needle (14): The partial structure for the chaperone-complexed EspA (15) revealed two long helices at the N and C termini of the molecule analogous to the flagellin D0 domain. These homologies suggest that MxiH may represent the minimum structural requirement to build a helical assembly for protein transport in these systems.The structural homology of MxiH to the Yersinia T3SS protein YscE (16) is even more striking because this protein is of similar size (≈8 kDa, although there is no significant primary sequence similarity) and possesses the same helix–turn–helix structure. YscE is absolutely required for secretion of Yersinia sp. T3SS effector proteins and has recently been proposed to be the chaperone for YscF, the Yersinia needle subunit (17, 18). The strong structural similarity between a needle subunit, MxiH, and a needle-subunit chaperone, YscE, suggests that, for these small needle subunits, the chaperone and needle-subunit interactions might mimic the monomer–monomer interactions seen within the assembled needle to block further polymerization, distinct from the interactions of the larger axial components with their chaperones [flagellin with FliS (19) and EspA with CesA (15)], where the chaperones effectively "cap" the termini of the subunits and so prevent polymerization via a very different set of interactions from those seen/predicted to occur in the assembled structure.
Burial of a MxiH monomer in the assembled A-needle.
The total accessible surface area (ASA), calculated by using AREAIMOL (20) of a MxiH monomer is 6,131 Å2, whereas the ASA of a MxiH monomer in the assembled A-needle (surrounded by six other monomers) is only 2,654 Å2. Thus, 57% of the surface of each monomer is buried upon needle assembly.1. Deane, J. E., Cordes, F. S., Roversi, P., Johnson, S., Kenjale, R., Picking, W. D., Picking, W. L., Lea, S. M. & Blocker, A. (2006) Acta Crystallogr. F 62, 302–305.
2. Derewenda, U., Mateja, A., Devedjiev, Y., Routzahn, K. M., Evdokimov, A. G., Derewenda, Z. S. & Waugh, D. S. (2004) Structure (London) 12, 301–306.
3. CCP4 (1994) Acta Crystallogr. D 50, 760–763.
4. Barrett, C. P., Hall, B. A. & Noble, M. E. (2004) Acta Crystallogr. D 60, 2280–2287.
5. Lindahl, E., Hess, B. & van der Spoel, D. (2001) J. Mol. Mod. 7, 306–317.
6. de La Fortelleqq, E. & Bricogne, G. (1997) Methods Enzymol. 276, 472–494.
7. Cowtan, K. (1994) Joint CCP4/ESF-EACBM Newslett. Protein Crystallogr. 31, 34–38.
8. Abrahams, J. P. & Leslie, A. G. (1996) Acta Crystallogr. D 52, 30–42.
9. McRee, D. E. (1999) J. Struct. Biol. 125, 156–165.
10. Blanc, E., Roversi, P., Vonrhein, C., Flensburg, C., Lea, S. M. & Bricogne, G. (2004) Acta Crystallogr. D 60, 2210–2221.
11. Perrakis, A., Morris, R. & Lamzin, V. S. (1999) Nat. Struct. Biol. 6, 458–463.
12. Kleywegt, G. J. & Jones, T. A. (1996) Acta Crystallogr. D 52, 826–828.
13. Yonekura, K., Maki-Yonekura, S. & Namba, K. (2003) Nature 424, 643–650.
14. Blocker, A., Komoriya, K. & Aizawa, S. (2003) Proc. Natl. Acad. Sci. USA 100, 3027–3030.
15. Yip, C. K., Finlay, B. B. & Strynadka, N. C. (2005) Nat. Struct. Mol. Biol. 12, 75–81.
16. Phan, J., Austin, B. P. & Waugh, D. S. (2005) Protein Sci. 14, 2759–2763.
17. Day, J. B., Guller, I. & Plano, G. V. (2000) Infect. Immun. 68, 6466–6471.
18. Quinaud, M., Chabert, J., Faudry, E., Neumann, E., Lemaire, D., Pastor, A., Elsen, S., Dessen, A. & Attree, I. (2005) J. Biol. Chem. 280, 36293–36300.
19. Evdokimov, A. G., Phan, J., Tropea, J. E., Routzahn, K. M., Peters, H. K., Pokross, M. & Waugh, D. S. (2003) Nat. Struct. Biol. 10, 789–793.
20. Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379–400.
21. Kenjale, R., Wilson, J., Zenk, S. F., Saurya, S., Picking, W. L., Picking, W. D. & Blocker, A. (2005) J. Biol. Chem. 280, 42929–42937.