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. 2009 May;16(5):477-85.
doi: 10.1038/nsmb.1599. Epub 2009 Apr 26.

Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout

Julie L Hodgkinson  1 , Ashley Horsley, David Stabat, Martha Simon, Steven Johnson, Paula C A da Fonseca, Edward P Morris, Joseph S Wall, Susan M Lea, Ariel J Blocker
Affiliations

Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout

Julie L Hodgkinson et al. Nat Struct Mol Biol. 2009 May.

Erratum in

  • Nat Struct Mol Biol. 2009 Aug;16(8):897

Abstract

Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic secretion machinery and a base that bridges both bacterial membranes, into which a hollow, external needle is embedded. When isolated, the latter two parts are termed the 'needle complex'. An incomplete understanding of the structure of the needle complex has hampered studies of T3SS function. To estimate the stoichiometry of its components, we measured the mass of its subdomains by scanning transmission electron microscopy (STEM). We determined subunit symmetries by analysis of top and side views within negatively stained samples in low-dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21-25-A resolution, three-dimensional reconstruction of the needle complex base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component.

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Figures

Figure 1
Figure 1
S. flexneri needle complexes. Electron micrograph of particles negatively stained with 2% w/v uranyl acetate. Examples of well-preserved particles are indicated (white stars). Note top views of base without needles (arrowed). Clearly damaged or tilted particles (black star) were excluded from the analysis. Scale bar represents 500Å.
Figure 2
Figure 2
STEM data collection and analysis. (a) Typical NC field used to collect data with picked particles (boxed). Long filaments are TMV used as internal calibration standard. (b) Average of boxed particles. c. Average mass map with background subtracted for picked particles in the example field shown (mass in kDa for each 2nm ]× 2nm area). Right hand column indicates mass sums (in kDa) for the horizontal slice of the mass map immediately to its left. Masses highlighted in dark grey, mid-grey, light grey, were assigned to needle, OMR and IMR respectively. The integrated mass sum of regions assigned to each component is shown at the bottom of the columns in assigned colour. Horizontal grey lines on mass map delineate range of slices summed. Scale bar is 100μm.
Figure 3
Figure 3
Image analysis of top views of the NC base. (a) Class averages containing 14, 21, 14, 19 and 12 images respectively. (b) Class averages of IMR (shown masked with the ring mask used for MSA for display purposes) show 24 subunits. Class 2 has a 24 fold overlay to illustrate this. These subunits often appear paired (arrow in a and b). (c) OMR only class averages (masked using MSA mask for display), although often 12 sub-units can be seen, indicated by dashed ring on image 1, these are more poorly resolved than for the IMR. Scale bar is 100Å.
Figure 4
Figure 4
Classification of NC side views. (a) Classification according to the IMR shows two sub-populations defined by the position of the “leg” portion (arrowed): closed legs, class 1 and open legs, class 2. (b) Surface representation of IMR models (C20, 24, 26 symmetries shown) used to check for symmetry variation in side views using composite models (see Supplementary Methods). (c) Selected class averages used for first C24 symmetry 3D reconstruction. Euler angles assigned to each class are indicated at the base of the figure. (d) Reprojections from the C24 3D reconstruction compare well with input class averages in c. Weak densities seen at extreme base end in class averages were highly disordered and did not reconstruct well and were therefore masked from final 3D reconstructions. Scale bar is 100Å.
Figure 5
Figure 5
3D reconstruction of NCs with C12 symmetry. (a) Surface representation. Labels on left hand side define structural regions in the map and numbers 1-8 on right hand side define points at which slices were taken for 5b. Conn.=connector, Le= legs. The bacterial (cytoplasmic) side of the NC, is defined as the base of the complex. (b) Slices through the 3D reconstructions at 1 (linker), 2 (IMR-shoulder), 3 (Shoulder-connector transition), 4 (Connector), 5 (Connector-OMR3 transition), 6 (OMR2), 7 (OMR2-1 transition) and 8 (OMR1). Arrow in b2 shows radial density spokes extending inward from between each pair of outer IMR sub-units. Slice 3 shows the top of IMR (large radius ring indicated) and start of connector (smaller radius ring indicated). Slices 3 and 5 show azimuthal tilts of connector sub-units. 12-fold symmetry is seen throughout the OMR rings (slices 6-8). More slices are shown in Supplementary Fig. 6. (c) Longitudinal cutaway of map reveals details of internal components with a distinct “socket-ring” (SR, blue arrow) with strong density modulations and 12 fold connections to the “socket-cup” (S/C) directly below it. Twelve spokes connect the socket-cup to the IMR (S). Two slender linkers (Li) connect the legs to the underside of the IMR shoulder. The top of the connector shows 12-fold connectivity with the underside of OMR3. N=needle. Hollow arrow on left side of connector indicates point at which structure cut for view in 5d (where black ring is cut-through volume). (d) Top view surface cut back to the socket-ring shows its 12-fold symmetry and domains with azimuthal tilt (blue arrow). The spokes connecting the outer IMR to the socket/cup are seen below this ring. (e) View of the NC base from the cytoplasmic side showing 12-fold symmetry of socket cup (black arrow). The 12-fold spokes display azimuthal tilting. Scale bar for a, c, d and e is 100Å. Scale bar for b is 100Å.
Figure 6
Figure 6
Possible fits of crystal structure of EscJ (MxiJ homolog) in the IMR/connector portions of the map. (a) Position of EscJ 24-mer (; red) fitted by automated methods (Molrep) to the outline of the C12 NC reconstruction (blue). (b) EscJ 24-mer placed manually in a position equivalent to Moraes et al. fit to 3D reconstruction of Marlovits et al.. In our map this placement was only possible via molecular replacement if that specific region of density was cut out of the map. (c) An EscJ 12-mer created by removing every other copy of the EscJ 24-mer, followed by a manual rotation of the subunits to best fit the spoke density. This 12-mer does position itself with Molrep in the full C12 NC reconstruction. The IM and OM (green) are shown for perspective. The membrane model used is POPC (palmitoyloleoylphosphatidylcholine). The IM is positioned at a mid-point of possible placements suggested in the literature. The up arrow indicates where the top of the top leaflet could reach and the down arrow indicates where the bottom of the bottom leaflet could reach. The needle model (70Å diameter, cyan; PDB code 2V6L14) is shown for scaling and was docked manually.
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References

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