1. Volume 25, Issue 9, Pages 1380-1390.e5 (September 2017)
A Janus-Faced IM30 Ring Involved in Thylakoid Membrane Fusion Is Assembled from IM30 Tetramers 
Michael Saur, Raoul Hennig, Phoebe Young, Kristiane Rusitzka, Nadja Hellmann, Jennifer Heidrich, Nina Morgner, Jürgen Markl, Dirk Schneider 
Structure 
Volume 25, Issue 9, Pages e5 (September 2017)
DOI: /j.str
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2. Structure 2017 25, 1380-1390.e5DOI: (10.1016/j.str.2017.07.001)
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3. Figure 1 IM30 Ring Structures Bound to Liposome Surfaces
(A) Upon addition of IM30 to liposomes, extra electron densities were found on membrane surfaces. Recently, this extra density has been superimposed with an IM30 ring having an 18-fold internal symmetry (modified from Hennig et al., 2015).
(B) In comparison to the previously published data, differently sized extra electron densities, which correspond to differently sized IM30 rings, were also observed on membrane surfaces.
The brackets illustrate the dimensions of an IM30 ring shown in (A) with 18-fold internal symmetry, and the shaded area marks the differences in the ring widths, i.e., differently sized IM30 rings. Scale bars, 25 nm.
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4. Figure 2
IM30 3D Structures
(A) Cross-section of an IM30 ring showing rails, spikes, and the ridge.
(B) Ring diameters increase linearly with ring size and ring symmetry.
(C) Differently sized IM30 rings displayed in side, tilted, and top views. The rings all have similar morphologies. Most notably, one side of the ring is more constricted (defined as the bottom of the ring) than the other (defined as top). Note that the ring height is constantly ∼14 nm.
Scale bars, 10 nm.
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5. Figure 3 IM30 Ring Segmentation
IM30 tetramers that form the basic ring building block could be either distributed as in (A) or in (B). Watershed segmentation (C) consistently yields (A) as the most probable distribution.
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6. Figure 4 Structural Comparison of IM30 WT and FERM Variants
(A and B) Transmission electron microscopy images of IM30 WT (A) and IM30_FERM (B). Scale bars, 200 nm.
(C and D) Analysis of IM30 WT and IM30_FERM via SEC using a Superose 12 column. IM30 WT (solid line) and IM30_FERM (dotted line) were analyzed after dialysis (C) or directly after elution of the sample from the Ni-NTA column (D).
(E) The mass of the IM30_FERM low-mass peak was estimated by SEC using a Superdex 200 column (elution volume 66.48 ± 0.35 mL; N = 3). The asterisks in (C, D, and E) mark the void volume (>660 kDa) of the columns as determined by thyroglobulin.
(F) The IM30_FERM molecular mass from (E) was calculated using the protein standards listed in the STAR Methods section.
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7. Figure 5 Mass Spectrometry of IM30 Low-Mass Complexes
The mass spectrum of IM30 low-mass complexes shows the presence of monomer, dimer, and tetramer, as well as a small amount of trimer. Oligomeric states are shown with their charge states indicated in superscript. Where m/z ratios lead to spectral overlap, both species are indicated.
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8. Figure 6 Organization of an IM30 Monomer within IM30 Rings
(A) Predicted localization of helices 1–7 (H1–7) within the IM30 sequence, highlighting the localization of the amino acid clusters, AAL, ELA, and FERM, analyzed in this study.
(B) Fitting of a PspA fragment crystal structure (PDB: 4WHE) into the IM30 ring reconstruction highlights superimpositions of defined features (arrowheads), namely the leftward kink at the bottom and the silhouette of the coiledcoil, as well as a bulge on the outer edge of the spike (red circle), which, in the heterologous expressed IM30, may only hold helix 1. The helix 2↔helix 3 connecting loop 2 is located at the ring bottom.
(C and D) Robetta-predicted full-length IM30 WT monomer structure (see STAR Methods). The red circle illustrates the N-terminal helix 1 (plus the N-terminal His-tag), the light blue circle shows the C-terminal domain and the black circle marks the loop 2 region. In (D) the predicted IM30 monomer structure is fitted into the IM30 ring in accordance with the fit shown in (B). Helix 1 (red circle) fills the distal bulge in the spike density. Helix 7 is likely situated in large unfilled densities at the top (blue circle). The negatively charged cluster of the helix 2↔helix 3 connecting loop 2 is localized at the ring bottom (black circle).
(E) Only four IM30 monomers properly filled the density of a basal ring “building block.” It is noteworthy that, while the tetramer can be defined as a basic building block for the IM30 ring, the exact assembly of the tetramer cannot be readily determined from our low-resolution structures.
(F) Displays a magnification of the negatively charged loop 2 region, including the well-conserved amino acids, as indicated in (B–D).
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9. Figure 7 IM30 Structure in the Presence of Mg2+
(A and B) TEM images of IM30 WT in the presence of (A) solely HEPES buffer (pH 7.6) or (B) with an additional 25 mM Mg2+. Double rings are indicated by white arrows. Scale bars, 200 nm (N = 3).
(C) Class sums of double-ring complexes in the presence of 25 mM Mg2+, showing ring-size independent formation of double-ring stacks.
(D) The hydrodynamic diameters of IM30 (■), IM30_AAL (▴) and IM30_ELA (▾) in the presence of increasing Mg2+ concentrations (0–25 mM) were analyzed by dynamic light scattering. Error bars: SD (N = 3).
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10. Figure 8 Membrane Interaction of the IM30 Variants AAL and ELA
(A) Laurdan spectra of pure PG liposomes (□) as well as of PG liposomes in the presence of IM30_ AAL (▴) and IM30_ELA (▾).
(B) Time-resolved membrane fusion in the presence of solely 7.5 mM Mg2+ (□) or after further addition of IM30 WT (■), IM30_AAL (▴), or IM30_ELA (▾), respectively. Error bars: SD (N = 3).
(C–F) Cryo-TEM analysis of liposomes in the presence of (C) solely 10 mM Mg2+ or of Mg2+ plus (D) IM30 WT, (E) IM30_AAL, or (F) IM30_ELA. Black arrows in (E and F) highlight some IM30 rings bound to liposomal surfaces. Scale bars, 200 nm.
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11. Figure 9 Model of IM30-Triggered Initiation of Membrane Fusion
An IM30 ring has two membrane binding surfaces. After binding to a membrane surface via the C-terminal IM30 domain (involving helix 7), the ring eventually binds via its bottom side to a second membrane. Due to IM30-induced membrane bending, the two membranes come into close proximity, eventually resulting in membrane fusion.
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