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. 2014 Aug 5;3:e03653. doi: 10.7554/eLife.03653

Figure 2. The structural organization of the LdcI–RavA cage.

(A) The RavA hexamer is represented as two triskelia. The pseudoatomic model of the RavA hexamer in the context of the LdcI–RavA complex is superimposed with the isolated RavA negative stain EM map (25 Å resolution, El Bakkouri et al., 2010) to show the conformational changes of the RavA legs induced by LdcI binding. (B–G) The RavA loop at the beginning of the LARA domain (amino acids 329–360) is shown as a broken line. (B) Schematics of LdcI–RavA interaction with RavA. (C) CryoEM map and pseudoatomic model of LdcI–RavA (11 Å resolution). This particular orientation of the complex illustrates the origins of the close-up views (E) and (G) surrounded with a solid rectangle and a broken line rectangle, respectively. (D) Top view of the cryoEM map and pseudoatomic model of the LdcI-LARA complex (7.5 Å resolution). (E) Close-up view of the LdcI–RavA complex (11 Å resolution) showing the LdcI-LARA interaction and arising from the bold rectangle in (C). (F) Close-up view of LdcI-LARA (7.5 Å resolution) in the same orientation as in (E). The higher resolution of this 3D reconstruction enables a more precise fitting of individual crystal structures. (G) Close-up view of the equatorial RavA–RavA interaction via the triple helical bundles and the foot–leg interaction (arising from the broken line rectangle in (C)).

DOI: http://dx.doi.org/10.7554/eLife.03653.007

Figure 2.

Figure 2—figure supplement 1. Conformation rearrangements of RavA induced by LdcI binding.

Figure 2—figure supplement 1.

(A) Pseudoatomic model of RavA from the LdcI–RavA complex superimposed with the negative stain EM map of RavA (25 Å resolution, El Bakkouri et al., 2010) to show three different conformations of the RavA leg: the bent conformation induced by the binding to LdcI outer ring in blue/dark blue, the extended conformation induced by the binding to LdcI inner ring in yellow/red, and the intermediate conformation induced by equatorial RavA–RavA binding in green/dark green. The reorientation of the LARA domains upon interaction with LdcI is clearly visible. (B–D) Orthogonal views of the RavA crystal structure monomer (gray, black) superimposed with each of the three different conformations of RavA from the LdcI–RavA pseudoatomic model. Colors as in (A).
DOI: http://dx.doi.org/10.7554/eLife.03653.008

Figure 2—figure supplement 2. CryoEM analysis of LdcI-LARA.

Figure 2—figure supplement 2.

(A) LdcI-LARA cryoEM micrograph displaying boxed particles. (B) Correspondence between projections of the 3D model and particle class averages showing the reliability of the 3D reconstruction. (C) Angular distribution of particles used for the final 3D reconstruction shown for the asymmetric unit. (D) Gold-standard FSC curves calculated for unmasked (blue) and soft shaped masked (red) maps, indicating the resolution of 8.8 Å and 7.5 Å respectively according to FSC = 0.143 criterion.
DOI: http://dx.doi.org/10.7554/eLife.03653.009

Figure 2—figure supplement 3. Insights into the LdcI-LARA interaction in LdcI–RavA and LdcI-LARA maps.

Figure 2—figure supplement 3.

(A–C) Superimposition of LdcI–RavA and LdcI-LARA maps highlighting the conservation of the LdcI-LARA structure. (A) Top view, (B) Close-up top view, (C) Close-up side view. (D and E) Fitting of the LdcI and LARA crystal structures into the LdcI-LARA map. The LARA domain is fitted as a rigid body, the LdcI monomer is fitted either (D) rigidly or (E) flexibly, which involved only subtle movements. (F) The difference between the rigid and the Flex-EM fit ot the LdcI monomer into the LdcI-LARA map presented in D and E is illustrated here in terms of the FSC curves between the corresponding model and the map (blue for the rigid fit and red for the Flex-EM model). (G) Quality of the Flex-EM model of the LdcI–RavA complex is illustrated by FSC curves indicating differences between the LdcI–RavA map and (i) dark blue–a rigid fit of the crystal structure of LdcI (Kanjee et al., 2011) and of the symmetric pseudoatomic model of the RavA hexamer (El Bakkouri et al., 2010) into it, (ii) red—the present Flex-EM model of the entire LdcI–RavA complex, (iii) green—the present Flex-EM model of the LdcI-LARA region (based on the LdcI-LARA map), (iv) light blue–the present Flex-EM model of the AAA+ domain core and the triple helical domains of RavA.
DOI: http://dx.doi.org/10.7554/eLife.03653.010

Figure 2—figure supplement 4. Mapping the interaction surfaces between RavA and LdcI.

Figure 2—figure supplement 4.

(A) List of designed mutations. Proteins, mutations, expected effects of mutations and aims of the experiments are indicated. Mutations are colored or highlighted with yellow stars, triangles and rectangles in a consistent way through the figure. (B) Sequence of the flexible region (residues 329–360) located between the second helix of the triple helical bundle (blue cylinder) and the first β-strand of the LARA domain of RavA (blue arrow). The designed mutations described in (A) are indicated. (C) Size exclusion chromatography analysis of the interaction between the different mutants probing LdcI–RavA interaction. The elution position of the molecular weight standards is shown on top. (D) Mutations presented in (C) localized on a cut-out of the 3D map and the pseudoatomic model of the LdcI-LARA complex. RavA mutations are highlighted as in (A) and LdcI mutations critical for the RavA binding (E634K and Y697S) are shown in green. LdcI is in light orange and the LARA domain in dark blue. The triple helical bundle of the entire RavA (light blue) is shown for clarity. (E) Size exclusion chromatography analysis of mutations probing the RavA–RavA interactions along the equator of the LdcI–RavA complex. (F) Mutations presented in (E) localized on a cut-out of the 3D map and the pseudoatomic model of the LdcI–RavA complex.
DOI: http://dx.doi.org/10.7554/eLife.03653.011

Figure 2—figure supplement 5. Comparison between triskelia of RavA and clathrin.

Figure 2—figure supplement 5.

(A) Two RavA triskelia are colored as in Figure 2. (B) A clathrin trikelion is colored in blue with the tip domain in dark blue. Since no 3D EM map of the 28 triskelia clathrin mini-coat that has approximately the same size as the LdcI–RavA complex is available, the structure of the bigger hexagonal D6 barrel (EMD-5119) is shown for comparison.
DOI: http://dx.doi.org/10.7554/eLife.03653.012