Figures and data in Structural basis for activation, assembly and membrane binding of ESCRT-III Snf7 filaments

Figures
Tables
Additional files

8 figures, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements

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X-ray Crystal Structure of Snf7^core

(A) The domain organization of Snf7. The core domain used for X-ray crystallography is shown in blue. (B) Overlay of ribbon and space-filling models of the X-ray crystal structure of Snf7^core. (C) Electrostatic surface potential of Snf7^core with positively charged regions in blue (+10kcal/e^-) and negatively charged regions in red (-10kcal/e^-). See also Table 1.

https://doi.org/10.7554/eLife.12548.003

Figure 1—figure supplement 1

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Protein purification of Snf7^core

(A) A superdex-200 gel filtration size exclusion chromatogram of Snf7^core. (B) A SDS-PAGE Coomassie brilliant blue staining of the gel filtration fractions corresponding to Snf7^core.

https://doi.org/10.7554/eLife.12548.004

Figure 1—figure supplement 2

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2Fc-Fo simulated-annealing composite-omit electron density maps contoured at 1.0σ of Snf7^core open conformations (A) A and (B) B.
https://doi.org/10.7554/eLife.12548.005

Figure 1—figure supplement 3

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Superimposing Snf7^core (blue) with (A) CHMP4B^α1-α2 (cyan) (PDB: 4ABM), with (B) CHMP3^α1-α4 (purple) (PDB: 3FRT), with (C) CHMP6^α1 (red) (PDB: 3HTU) Snf7^core, and with (D) IST1^α1-α6 (grey) (PDB: 3FRR).
https://doi.org/10.7554/eLife.12548.006

Figure 2 with 3 supplements

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Conformational Rearrangement of Snf7

(**A–B**) Ribbon diagrams of (A) a homology model of closed Snf7^core (Henne et al., 2012) and (B) the X-ray crystal structure of open Snf7^core. (C) A close-up view of the side chain interaction between Gln90 and Met130. (D) Western blotting and subcellular fractionation of *snf7*Δ yeast exogenously expressing *SNF7* or *snf7^{Q90C M130C}*with and without copper(II) 1,10-phenanthroline. (E) Schematic showing closed and open Snf7^core with cysteines (red dots) before and after SDS-denaturing. (F) Snf7 site-directed spin-labeling with MTSL (red). (**G–H**) Distance between Glu88 and His118 of (G) closed and (H) open Snf7 shown in ribbon. (I and K) Time domain signals and distance distributions from DEER spectroscopy of (I) Snf7^{R52E E88C H118C} in solution, and simulated closed and open Snf7^{core E88C H118C} using *MMM*, and (K) Snf7^{R52E E88C H118C}: Snf7^R52E (1:0, 1:1, 1:2, and 1:8) with liposomes. (J) Schematic showing liposome sedimentation for DEER. MTSL-labeled Snf7 proteins (blue oval) and liposomes (grey circle).

https://doi.org/10.7554/eLife.12548.008

Figure 2—figure supplement 1

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Conceptual model for the Mup1-pHluorin MVB sorting assay.
https://doi.org/10.7554/eLife.12548.009

Figure 2—figure supplement 2

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Sequence alignments of Snf7 α2 and α4, with conserved Gln90 and Met130 shown in red, and quantitative MVB sorting data for *snf7*Δ yeast exogenously expressing *SNF7, snf7^Q90C, snf7^M130C*, and *snf7^{Q90C M130C}*.

Error bars represent standard deviations.

https://doi.org/10.7554/eLife.12548.010

Figure 2—figure supplement 3

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Time domain signals and distance distributions from DEER spectroscopy of full-length Snf7^{R52E E88C H118C}, Snf7^{R52E H118C G140C} and Snf7^{R52E E88C G140C}.

(A) Ribbon models of closed and open Snf7^core showing inter-residue distances between E88, H118 and G140. (**B–D**) Time domain signals and distance distributions from DEER spectroscopy of (B) full-length Snf7^{R52E E88C H118C}, and full-length Snf7^{R52E E88C H118C:}Snf7^R52E (1:1) in solution, (C) full-length Snf7^{R52E H118C G140C}in solution and simulated closed and open Snf7^{core H118C G140C} using *MMM*, and (D) full-length Snf7^{R52E E88C G140C} in solution and simulated closed and open Snf7^{core E88C G140C} using *MMM*. Blue shaded portions of the distributions indicate distance ranges that can be attributed to open and closed conformations.

https://doi.org/10.7554/eLife.12548.011

Figure 3 with 4 supplements

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Membrane-bound Snf7 Protofilament with ~30 Å Periodicity

(A) Overlay of ribbon and space-filling models of a 7-mer Snf7 protofilament with measured dimensions. (B and D) Time domain signals and distance distributions from DEER spectroscopy of (B) full-length Snf7^{R52E T20C}, Snf7^{R52E K35C}, and Snf7^{R52E E88C} with liposomes, (D) full-length Snf7^{R52E K60C}, Snf7^{R52E H118C}, and Snf7^{R52E G140C} with liposomes. (C and E) Schematic showing the spin label positions in a Snf7 protofilament.

https://doi.org/10.7554/eLife.12548.012

Figure 3—figure supplement 1

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Time domain signals and distance distributions from DEER spectroscopy of full-length Snf7^{R52E K60C A66C} in solution and full-length Snf7^{R52E K60C A66C}: Snf7^R52E (1:0, 1:2) with liposomes, and schematic showing the locations of the spin label positions in a Snf7 protofilament.
https://doi.org/10.7554/eLife.12548.013

Figure 3—figure supplement 2

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Time domain signals and distance distributions from DEER spectroscopy of full-length Snf7^{R52E E88C H118C}: Snf7^R52E (1:0, 1:2.5, 1:4, 1:8) with liposomes and simulated Snf7^{core E88C H118C}: Snf7^core (1:0, 1: ∞) polymers using *MMM*, and schematic showing the locations of the spin label positions in a Snf7 protofilament.

The full-length Snf7^{R52E E88C H118C}: Snf7^R52E (1:0 and 1:8) with liposomes datasets are re-plotted from Figure 2K as shown in fine lines.

https://doi.org/10.7554/eLife.12548.014

Figure 3—figure supplement 3

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Quantitative MVB sorting data for *snf7*Δ yeast exogenously expressing *SNF7, snf7^T20C, snf7^K35C, snf7^K60C, snf7^E88C, snf7^H118C, snf7^G140C, snf7^{K60C A66C}* and *snf7^{E88C H118C}*.

Error bars represent standard deviations.

https://doi.org/10.7554/eLife.12548.015

Figure 3—figure supplement 4

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Representative TEM images of recombinant full-length Snf7^{R52E K35C}, Snf7^{R52E E88C}, Snf7^{R52E K60C A66C}, and Snf7^{R52E E88C H118C} labeled with MTSL.

Scale bars, 200 nm.

https://doi.org/10.7554/eLife.12548.016

Figure 4 with 5 supplements

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Hydrophobic and Electrostatic Interactions in a Snf7 Filament

(**A–B**) Ribbon models of a Snf7 protofilament. The hydrophobic protein interface is shown in black dash-line and the electrostatic interface in grey dash-dot line. (**C–D**) Close-up views of the hydrophobic interface between α2/3ⁱ and α3ⁱ⁺¹ and the electrostatic interface between α1ⁱ and α2/3ⁱ⁺¹. Protomer (i) shown in yellow and protomer (i+1) in red. (E) Conceptual model for the Mup1-pHluorin MVB sorting assay. Vacuole (v). (F) Quantitative MVB sorting data for *snf7*Δ yeast exogenously expressing empty vector, *SNF7, snf7^L121D, snf7^I117E, snf7^M114E, snf7^M107E, snf7^T103E, snf7^L99K, snf7^M104E, snf7^L101E, snf7^A97K, snf7^I94E, snf7^Q90K, snf7^M87E,* and *snf7^T83E*. Error bars represent standard deviations. (G) Quantitative MVB sorting data for *snf7*Δ yeast exogenously expressing empty vectors, *SNF7, snf7^{R25E H29E K36E}*and empty vector, empty vector and *snf7^{E95K E102K E109K}*, and *snf7^{R25E H29E K36E}* and *snf7^{E95K E102K E109K}*. Error bars represent standard deviations. (H) Representative TEM images of recombinant full-length Snf7^R52E, Snf7^{R52E Q90K}, Snf7^{R52E M107E}, Snf7^{R52E R25E H29E K36E}, Snf7^{R52E E95K E102K E109K}, and Snf7^{R52E R25E H29E K36E} and Snf7^{R52E E95K E102K E109K} (1:1). Scale bars, 200 nm.

https://doi.org/10.7554/eLife.12548.017

Figure 4—figure supplement 1

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Hydrophobic Interface Mutant Analysis.

(A) Representative TEM images of recombinant full-length Snf7^R52EI94E and Snf7^R52EM114E. Scale bars, 200nm. (B) Superdex-200 gel filtration size exclusion chromatograms of Snf7^R52E, Snf7^R52EI94E and Snf7^{R52E M107E}. Related to Figure 4H.

https://doi.org/10.7554/eLife.12548.018

Figure 4—figure supplement 2

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Western blotting analyses of *snf7*Δ yeast expressing *SNF7, snf7^L121D, snf7^I117E, snf7^M114E, snf7^M107E, snf7^T103E*, and *snf7^L99K*, and *SNF7, snf7^M104E, snf7^L101E, snf7^A97K, snf7^I94E, snf7^Q90K, snf7^M87E*, and *snf7^T83E*.

G6PDH used as loading controls.

Sequence analyses of Snf7 α2/3 with conserved residues shown in gold and dark red.

https://doi.org/10.7554/eLife.12548.019

Figure 4—figure supplement 3

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Quantitative MVB sorting data for *snf7*Δ yeast exogenously expressing empty vector, *SNF7, snf7^R25E, snf7^H29E, snf7^K36E, snf7^E95K, snf7^E102K, and snf7^E109K*, and empty vector, *SNF7, snf7^{R25E K36E}*and *vector, vector* and *snf7^{E95K E109K}, snf7^{R25E K36E}*and *snf7^{E95K E109K}*.

Error bars represent standard deviations.

https://doi.org/10.7554/eLife.12548.020

Figure 4—figure supplement 4

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Western blotting analyses of *snf7*Δ yeast expressing *SNF7, snf7^{R25E H29E K36E}*, and *snf7^{E95K E102K E109K}*.

Sequence analyses of Snf7 α2/3 with conserved residues shown in gold and dark red.

https://doi.org/10.7554/eLife.12548.021

Figure 4—figure supplement 5

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Western blotting analyses of *ex vivo* P13 fractions BMOE crosslinking by Snf7^K35C with Snf7^K60C, Snf7^A63C, Snf7^K69C, Snf7^Q75C, Snf7^E81C, Snf7^E88C, Snf7^E95C, and Snf7^E102C.
https://doi.org/10.7554/eLife.12548.022

Figure 5

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Electrostatic Protein-membrane Interactions in a Snf7 Filament

(A) A Snf7 protofilament in ribbons placed on a lipid membrane in spheres (grey) (Heller et al., 1993). (B) Electrostatic surface potential showing the membrane interacting surface of a Snf7protofilament with positively charged regions in blue (+10kcal/e^-) and negatively charged regions in red (-10kcal/e^-).

https://doi.org/10.7554/eLife.12548.023

Figure 6 with 5 supplements

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Snf7 α4 in Inter-Filament Interactions

(**A–B**) Snf7^core conformations A (green) and B (blue) superimposed. (B) 90^° rotation and superimposing with a closed CHMP3 (purple) using its α3 as a reference. (C) Overlay of ribbon and space-filling models of the Snf7^core crystal packing of the open conformation A. The dash-line box represents the interfilament contacts. Arrows represent inter-protofilament orientations. (**D–E**) Close-up views of the hydrophobic interface between α1/2ⁱ (blue) and α4^j (yellow) of open conformations (D) A and (E) B. (F) Quantitative MVB sorting data for *snf7*Δ yeast exogenously expressing empty vector, *SNF7, snf7^V126E, snf7^M130E, snf7^I133E, snf7^A51E, snf7^L55E*, and *snf7^L67E*. Error bars represent standard deviations. See also Table 1.

https://doi.org/10.7554/eLife.12548.024

Figure 6—figure supplement 1

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Representative TEM images of recombinant full-length Snf7^{R52E V126E} and Snf7^{R52E I133E}.

Scale bars, 200nm.

https://doi.org/10.7554/eLife.12548.025

Figure 6—figure supplement 2

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Western blotting analyses of *snf7*Δ yeast expressing *SNF7, snf7^A51E, snf7^L55E, snf7^L67E, snf7^V126E, snf7^M130E*, and *snf7^I133E*.

G6PDH as a loading control.

Sequence analyses of Snf7 α1/2 and α4 with conserved residues shown in blue or gold.

https://doi.org/10.7554/eLife.12548.026

Figure 6—figure supplement 3

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Quantitative MVB sorting data for *snf7*Δ yeast exogenously expressing *SNF7, snf7^E102P, snf7^N59P*, and *snf7^L121P*.

Error bars represent standard deviations. Overlay ribbon models of (upper right) closed (purple) and open (blue) Snf7^core with Glu102 shown in sticks, and (lower right) open conformation A (green) and B (blue) with Asn59 and Leu121 shown in sticks. Arrows represent conformational rearrangements.

https://doi.org/10.7554/eLife.12548.027

Figure 6—figure supplement 4

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An overlay of ribbon and space-filling models of the Snf7^core crystal packing of the open conformation B.

The dash-line box represent the interfilament contacts shown in Figure 6E.

https://doi.org/10.7554/eLife.12548.028

Figure 6—figure supplement 5

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Superimposing of Snf7^core subunit (i) (blue), (j) (yellow) and CHMP3^α1-α5 (purple) of open conformations A (upper) and B (lower).
https://doi.org/10.7554/eLife.12548.029

Figure 7 with 3 supplements

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Models of Snf7 activation, polymer assembly and membrane remodeling

(A) Space-filling CONSURF models with high conservation (purple) and low conservation (cyan). Interacting protomers shown in ribbon (blue). Seven conserved regions with assigned functions labeled. Gray arrows indicate the flexibility of α4. (B) Speculative cartoons illustrating four stages in ESCRT-mediated vesicle budding. (C) Space-filling models and schematic cartoons of Snf7^core in closed and open states with membrane (grey). (D) Space-filling and close-up ribbon models of a 25-mer Snf7 single filament with membrane. (E) Space-filling and close-up ribbon models of a 23-mer Snf7 normal mode analysis filament with membrane (grey). (F) Schematic of a Snf7 homo-polymer in the neck of a nascent ILV with positive and negative membrane curvatures.

https://doi.org/10.7554/eLife.12548.030

Figure 7—figure supplement 1

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Alignment of Snf7^core protein sequences from *Saccharomyces cerevisiae (Sc), Homo sapiens (Hs), Mus musculus (Mm), Xenopus laevis (Xl), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Schizosaccharomyces pombe (Sp*) and *Lokiarchea* (Spang et al., 2015).
https://doi.org/10.7554/eLife.12548.031

Figure 7—figure supplement 2

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A ribbon model of a supercomplex of Vps25-Vps20-Snf7.

The first Snf7’s α1 was used for superimposing with the Vps20 α1 (Im et al., 2009) (PDB: 3HTU) for molecular docking.

https://doi.org/10.7554/eLife.12548.032

Figure 7—figure supplement 3

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Architectures of Snf7 protofilaments

(A) A representative TEM image of recombinant Snf7^R52E (left) and a space-filling model of a 61-mer Snf7^α1-3 straight filament shown in the same scale (right). (B) A representative TEM image of recombinant full-length Snf7^R52E, Vps24 and Vps2 (2:1:1) (left), and space-filling and close-up view of ribbon models of a 97-mer Snf7^α1-3 superhelix generated by normal mode analysis with measured dimensions (right). TEM scale bars, 50nm.

https://doi.org/10.7554/eLife.12548.033

Author response image 1

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Size Exclusion Chromatograms of full-length Snf7^R52E, Snf7^{R52E I94E} and Snf7^{R52E M107E} by Superdex-200 gel filtration.
https://doi.org/10.7554/eLife.12548.035

Tables

Table 1

Crystallographic Data Collection and Refinement Statistics

https://doi.org/10.7554/eLife.12548.007

	Snf7^core
	Conformation A	Conformation B
Wavelength (Å)	0.978	0.978
Resolution range (Å)	50 - 2.4 (2.49 - 2.40)	50 - 1.6 (1.6 - 1.55)
Space group	P2₁	P2₁
Unit cell	a = 29.5Å b = 52.2Å c = 54.5Å α = 90^°β = 97.5^°γ = 90^°	a = 29.9Å b = 46.2Å c = 44.6Å α = 90^°β = 98.5^°γ = 90^°
Total reflections	23263 (1946)	73723 (6034)
Unique reflections	6376 (612)	16849 (1581)
Multiplicity	3.6 (3.2)	4.4 (3.8)
Completeness (%)	97.99 (93.72)	95.77 (90.65)
Mean I/sigma(I)	8.04 (2.91)	8.85 (1.35)
Wilson B-factor	54.03	25.39
R_merge	0.0884 (0.249)	0.0782 (0.997)
CC_1/2	0.988 (0.968)	0.995 (0.590)
CC*	0.997 (0.992)	0.999 (0.861)
R_work	0.259 (0.398)	0.210 (0.330)
R_free	0.262 (0.533)	0.225 (0.356)
Number of non-hydrogen atoms	982	1097
macromolecules	975	992
water	7	105
Protein residues	123	125
RMS(bonds) (Å)	0.015	0.006
RMS(angles) (^o)	1.24	0.81
Ramachandran favored (%)	95	99
Ramachandran outliers (%)	1.7	0
Clashscore	21.56	9.9
Average B-factor	91.1	39.7
macromolecules	91.2	38.9
solvent	69.2	47.2

Additional files

Supplementary file 1

Plasmids and yeast strains used in this study.

A list of plasmids for Saccharomyces cerevisiae expression, Escherichia coli expression for protein purification, and Saccharomyces cerevisiae strains.: https://doi.org/10.7554/eLife.12548.034
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Shaogeng Tang
W Mike Henne
Peter P Borbat
Nicholas J Buchkovich
Jack H Freed
Yuxin Mao
J Christopher Fromme
Scott D Emr

(2015)

Structural basis for activation, assembly and membrane binding of ESCRT-III Snf7 filaments

eLife 4:e12548.

https://doi.org/10.7554/eLife.12548

Figures

X-ray Crystal Structure of Snf7^core

Protein purification of Snf7^core

2Fc-Fo simulated-annealing composite-omit electron density maps contoured at 1.0σ of Snf7^core open conformations (A) A and (B) B.

Superimposing Snf7^core (blue) with (A) CHMP4B^α1-α2 (cyan) (PDB: 4ABM), with (B) CHMP3^α1-α4 (purple) (PDB: 3FRT), with (C) CHMP6^α1 (red) (PDB: 3HTU) Snf7^core, and with (D) IST1^α1-α6 (grey) (PDB: 3FRR).

Conformational Rearrangement of Snf7

Conceptual model for the Mup1-pHluorin MVB sorting assay.

Sequence alignments of Snf7 α2 and α4, with conserved Gln90 and Met130 shown in red, and quantitative MVB sorting data for snf7Δ yeast exogenously expressing SNF7, snf7^Q90C, snf7^M130C, and snf7^{Q90C M130C}.

Time domain signals and distance distributions from DEER spectroscopy of full-length Snf7^{R52E E88C H118C}, Snf7^{R52E H118C G140C} and Snf7^{R52E E88C G140C}.

Membrane-bound Snf7 Protofilament with ~30 Å Periodicity

Time domain signals and distance distributions from DEER spectroscopy of full-length Snf7^{R52E K60C A66C} in solution and full-length Snf7^{R52E K60C A66C}: Snf7^R52E (1:0, 1:2) with liposomes, and schematic showing the locations of the spin label positions in a Snf7 protofilament.

Quantitative MVB sorting data for snf7Δ yeast exogenously expressing SNF7, snf7^T20C, snf7^K35C, snf7^K60C, snf7^E88C, snf7^H118C, snf7^G140C, snf7^{K60C A66C} and snf7^{E88C H118C}.

Representative TEM images of recombinant full-length Snf7^{R52E K35C}, Snf7^{R52E E88C}, Snf7^{R52E K60C A66C}, and Snf7^{R52E E88C H118C} labeled with MTSL.

Hydrophobic and Electrostatic Interactions in a Snf7 Filament

Hydrophobic Interface Mutant Analysis.

Western blotting analyses of snf7Δ yeast expressing SNF7, snf7^L121D, snf7^I117E, snf7^M114E, snf7^M107E, snf7^T103E, and snf7^L99K, and SNF7, snf7^M104E, snf7^L101E, snf7^A97K, snf7^I94E, snf7^Q90K, snf7^M87E, and snf7^T83E.

Quantitative MVB sorting data for snf7Δ yeast exogenously expressing empty vector, SNF7, snf7^R25E, snf7^H29E, snf7^K36E, snf7^E95K, snf7^E102K, and snf7^E109K, and empty vector, SNF7, snf7^{R25E K36E}and vector, vector and snf7^{E95K E109K}, snf7^{R25E K36E}and snf7^{E95K E109K}.

Western blotting analyses of snf7Δ yeast expressing SNF7, snf7^{R25E H29E K36E}, and snf7^{E95K E102K E109K}.

Western blotting analyses of ex vivo P13 fractions BMOE crosslinking by Snf7^K35C with Snf7^K60C, Snf7^A63C, Snf7^K69C, Snf7^Q75C, Snf7^E81C, Snf7^E88C, Snf7^E95C, and Snf7^E102C.

Electrostatic Protein-membrane Interactions in a Snf7 Filament

Snf7 α4 in Inter-Filament Interactions

Representative TEM images of recombinant full-length Snf7^{R52E V126E} and Snf7^{R52E I133E}.

Western blotting analyses of snf7Δ yeast expressing SNF7, snf7^A51E, snf7^L55E, snf7^L67E, snf7^V126E, snf7^M130E, and snf7^I133E.

Quantitative MVB sorting data for snf7Δ yeast exogenously expressing SNF7, snf7^E102P, snf7^N59P, and snf7^L121P.

An overlay of ribbon and space-filling models of the Snf7^core crystal packing of the open conformation B.

Superimposing of Snf7^core subunit (i) (blue), (j) (yellow) and CHMP3^α1-α5 (purple) of open conformations A (upper) and B (lower).

Models of Snf7 activation, polymer assembly and membrane remodeling

Alignment of Snf7^core protein sequences from Saccharomyces cerevisiae (Sc), Homo sapiens (Hs), Mus musculus (Mm), Xenopus laevis (Xl), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Schizosaccharomyces pombe (Sp) and Lokiarchea (Spang et al., 2015).

A ribbon model of a supercomplex of Vps25-Vps20-Snf7.

Architectures of Snf7 protofilaments

Size Exclusion Chromatograms of full-length Snf7^R52E, Snf7^{R52E I94E} and Snf7^{R52E M107E} by Superdex-200 gel filtration.

Tables

Additional files

Supplementary file 1

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X-ray Crystal Structure of Snf7core

Protein purification of Snf7core

2Fc-Fo simulated-annealing composite-omit electron density maps contoured at 1.0σ of Snf7core open conformations (A) A and (B) B.

Superimposing Snf7core (blue) with (A) CHMP4Bα1-α2 (cyan) (PDB: 4ABM), with (B) CHMP3α1-α4 (purple) (PDB: 3FRT), with (C) CHMP6α1 (red) (PDB: 3HTU) Snf7core, and with (D) IST1α1-α6 (grey) (PDB: 3FRR).

Conformational Rearrangement of Snf7

Conceptual model for the Mup1-pHluorin MVB sorting assay.

Sequence alignments of Snf7 α2 and α4, with conserved Gln90 and Met130 shown in red, and quantitative MVB sorting data for snf7Δ yeast exogenously expressing SNF7, snf7Q90C, snf7M130C, and snf7Q90C M130C.

Time domain signals and distance distributions from DEER spectroscopy of full-length Snf7R52E E88C H118C, Snf7R52E H118C G140C and Snf7R52E E88C G140C.

Membrane-bound Snf7 Protofilament with ~30 Å Periodicity

Time domain signals and distance distributions from DEER spectroscopy of full-length Snf7R52E K60C A66C in solution and full-length Snf7R52E K60C A66C: Snf7R52E (1:0, 1:2) with liposomes, and schematic showing the locations of the spin label positions in a Snf7 protofilament.

Quantitative MVB sorting data for snf7Δ yeast exogenously expressing SNF7, snf7T20C, snf7K35C, snf7K60C, snf7E88C, snf7H118C, snf7G140C, snf7K60C A66C and snf7E88C H118C.

Representative TEM images of recombinant full-length Snf7R52E K35C, Snf7R52E E88C, Snf7R52E K60C A66C, and Snf7R52E E88C H118C labeled with MTSL.

Hydrophobic and Electrostatic Interactions in a Snf7 Filament

Hydrophobic Interface Mutant Analysis.

Western blotting analyses of snf7Δ yeast expressing SNF7, snf7L121D, snf7I117E, snf7M114E, snf7M107E, snf7T103E, and snf7L99K, and SNF7, snf7M104E, snf7L101E, snf7A97K, snf7I94E, snf7Q90K, snf7M87E, and snf7T83E.

Quantitative MVB sorting data for snf7Δ yeast exogenously expressing empty vector, SNF7, snf7R25E, snf7H29E, snf7K36E, snf7E95K, snf7E102K, and snf7E109K, and empty vector, SNF7, snf7R25E K36Eand vector, vector and snf7E95K E109K, snf7R25E K36Eand snf7E95K E109K.

Western blotting analyses of snf7Δ yeast expressing SNF7, snf7R25E H29E K36E, and snf7E95K E102K E109K.

Western blotting analyses of ex vivo P13 fractions BMOE crosslinking by Snf7K35C with Snf7K60C, Snf7A63C, Snf7K69C, Snf7Q75C, Snf7E81C, Snf7E88C, Snf7E95C, and Snf7E102C.

Electrostatic Protein-membrane Interactions in a Snf7 Filament

Snf7 α4 in Inter-Filament Interactions

Representative TEM images of recombinant full-length Snf7R52E V126E and Snf7R52E I133E.

Western blotting analyses of snf7Δ yeast expressing SNF7, snf7A51E, snf7L55E, snf7L67E, snf7V126E, snf7M130E, and snf7I133E.

Quantitative MVB sorting data for snf7Δ yeast exogenously expressing SNF7, snf7E102P, snf7N59P, and snf7L121P.

An overlay of ribbon and space-filling models of the Snf7core crystal packing of the open conformation B.

Superimposing of Snf7core subunit (i) (blue), (j) (yellow) and CHMP3α1-α5 (purple) of open conformations A (upper) and B (lower).

Models of Snf7 activation, polymer assembly and membrane remodeling

Alignment of Snf7core protein sequences from Saccharomyces cerevisiae (Sc), Homo sapiens (Hs), Mus musculus (Mm), Xenopus laevis (Xl), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Schizosaccharomyces pombe (Sp) and Lokiarchea (Spang et al., 2015).

A ribbon model of a supercomplex of Vps25-Vps20-Snf7.

Architectures of Snf7 protofilaments

Size Exclusion Chromatograms of full-length Snf7R52E, Snf7R52E I94E and Snf7R52E M107E by Superdex-200 gel filtration.

Supplementary file 1

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X-ray Crystal Structure of Snf7^core

Protein purification of Snf7^core

2Fc-Fo simulated-annealing composite-omit electron density maps contoured at 1.0σ of Snf7^core open conformations (A) A and (B) B.

Superimposing Snf7^core (blue) with (A) CHMP4B^α1-α2 (cyan) (PDB: 4ABM), with (B) CHMP3^α1-α4 (purple) (PDB: 3FRT), with (C) CHMP6^α1 (red) (PDB: 3HTU) Snf7^core, and with (D) IST1^α1-α6 (grey) (PDB: 3FRR).

Sequence alignments of Snf7 α2 and α4, with conserved Gln90 and Met130 shown in red, and quantitative MVB sorting data for snf7Δ yeast exogenously expressing SNF7, snf7^Q90C, snf7^M130C, and snf7^{Q90C M130C}.

Time domain signals and distance distributions from DEER spectroscopy of full-length Snf7^{R52E E88C H118C}, Snf7^{R52E H118C G140C} and Snf7^{R52E E88C G140C}.

Time domain signals and distance distributions from DEER spectroscopy of full-length Snf7^{R52E K60C A66C} in solution and full-length Snf7^{R52E K60C A66C}: Snf7^R52E (1:0, 1:2) with liposomes, and schematic showing the locations of the spin label positions in a Snf7 protofilament.

Quantitative MVB sorting data for snf7Δ yeast exogenously expressing SNF7, snf7^T20C, snf7^K35C, snf7^K60C, snf7^E88C, snf7^H118C, snf7^G140C, snf7^{K60C A66C} and snf7^{E88C H118C}.

Representative TEM images of recombinant full-length Snf7^{R52E K35C}, Snf7^{R52E E88C}, Snf7^{R52E K60C A66C}, and Snf7^{R52E E88C H118C} labeled with MTSL.

Western blotting analyses of snf7Δ yeast expressing SNF7, snf7^L121D, snf7^I117E, snf7^M114E, snf7^M107E, snf7^T103E, and snf7^L99K, and SNF7, snf7^M104E, snf7^L101E, snf7^A97K, snf7^I94E, snf7^Q90K, snf7^M87E, and snf7^T83E.

Quantitative MVB sorting data for snf7Δ yeast exogenously expressing empty vector, SNF7, snf7^R25E, snf7^H29E, snf7^K36E, snf7^E95K, snf7^E102K, and snf7^E109K, and empty vector, SNF7, snf7^{R25E K36E}and vector, vector and snf7^{E95K E109K}, snf7^{R25E K36E}and snf7^{E95K E109K}.

Western blotting analyses of snf7Δ yeast expressing SNF7, snf7^{R25E H29E K36E}, and snf7^{E95K E102K E109K}.

Western blotting analyses of ex vivo P13 fractions BMOE crosslinking by Snf7^K35C with Snf7^K60C, Snf7^A63C, Snf7^K69C, Snf7^Q75C, Snf7^E81C, Snf7^E88C, Snf7^E95C, and Snf7^E102C.

Representative TEM images of recombinant full-length Snf7^{R52E V126E} and Snf7^{R52E I133E}.

Western blotting analyses of snf7Δ yeast expressing SNF7, snf7^A51E, snf7^L55E, snf7^L67E, snf7^V126E, snf7^M130E, and snf7^I133E.

Quantitative MVB sorting data for snf7Δ yeast exogenously expressing SNF7, snf7^E102P, snf7^N59P, and snf7^L121P.

An overlay of ribbon and space-filling models of the Snf7^core crystal packing of the open conformation B.

Superimposing of Snf7^core subunit (i) (blue), (j) (yellow) and CHMP3^α1-α5 (purple) of open conformations A (upper) and B (lower).

Alignment of Snf7^core protein sequences from Saccharomyces cerevisiae (Sc), Homo sapiens (Hs), Mus musculus (Mm), Xenopus laevis (Xl), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Schizosaccharomyces pombe (Sp) and Lokiarchea (Spang et al., 2015).

Size Exclusion Chromatograms of full-length Snf7^R52E, Snf7^{R52E I94E} and Snf7^{R52E M107E} by Superdex-200 gel filtration.