1. Structural Basis of Atg8 Activation by a Homodimeric E1, Atg7
Nobuo N. Noda, Kenji Satoo, Yuko Fujioka, Hiroyuki Kumeta, Kenji Ogura, Hitoshi Nakatogawa, Yoshinori Ohsumi, Fuyuhiko Inagaki 
Molecular Cell 
Volume 44, Issue 3, Pages (November 2011)
DOI: /j.molcel
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2. Molecular Cell 2011 44, 462-475DOI: (10.1016/j.molcel.2011.08.035)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 Overall Structure of Atg7
(A) Summary of the Atg7 constructs used for structural studies in this paper.
(B) Ribbon representation of the monomer structure of Atg7(1–613). NTD, Linker, AD, and ECTD of Atg7 are colored cyan, orange, green, and yellow, respectively. α helices and β strands are labeled α and β with numbers, and N and C termini are labeled N and C, respectively. All the structural models in this manuscript were prepared with PyMOL (DeLano, 2002).
(C) Ribbon representation of the homodimer structure of Atg7(1–613). One molecule is colored as in (B), while the other is colored gray. The lower panel is obtained by 90° rotation of the upper panel around the horizontal axis.
(D) In vitro pull-down assay between GST-fused Atg7s and Atg3 or Atg8. Eluted proteins were analyzed by SDS-PAGE and detected with Coomassie brilliant blue (CBB) staining.
(E) In vitro activation of Atg8 by Atg7. Full-length Atg7 or Atg7CTD was incubated with Atg8 and MgATP and was subjected to SDS-PAGE followed by CBB staining.
See also Figure S1.
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4. Figure 2 Characterization of the Crucial Residues in Atg7NTD
(A) Electrostatic surface potentials calculated for the surface of the Atg7NTD structure with PyMOL. Blue and red indicate positive and negative potentials, respectively. Highly basic surface is circled with a cyan, broken circle. Residues subjected to mutational analysis are labeled.
(B) In vitro pull-down assay between GST-fused Atg7 mutants and Atg3. Input and eluted proteins were subjected to SDS-PAGE followed by CBB staining. Asterisks indicate nonspecific bands. Atg3 binding was calculated by dividing the ratio of eluted Atg3 and GST-Atg7 mutant amounts with that of eluted Atg3 and wild-type GST-Atg7 amounts. The values and the error bars in (B) and (C) are the means and the standard deviations of three independent experiments, respectively.
(C) Single-turnover assay of Atg3∼Atg8 thioester intermediate formation. Atg3∼Atg8 formation was calculated by dividing the amount of Atg3∼Atg8 intermediate with that of free Atg3.
See also Figure S2 for multiple sequence alignment of Atg7 homologs.
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5. Figure 3 Crystal Structure of Atg7CTD-Atg8 Complex
(A) Ribbon representation of the overall structure of Atg7CTD-Atg8 complex. Bound ATP is shown with a stick model. Atg7 is colored as in Figure 1B, Atg8 is colored salmon pink, and ATP is colored red for one complex, while another complex is colored gray.
(B) Stereo representation of the detailed interaction between Atg7 and Atg8. Coloring is as in (A). The C-terminal tail of Atg8 and the side chains of the residues involved in Atg7-Atg8 interaction are shown with stick models. Possible hydrophilic interactions are shown with broken lines.
(C) Surface representation of Atg7CTD bound to Atg8 (left) and free Atg7 (right). The bound Atg8 and ATP are shown with ribbon and stick models, respectively. Atg7CL is colored blue and black for Atg8-bound Atg7CTD and free Atg7, respectively. Two figures are shown in the same orientation.
(D) Superimposition of Atg7CTD bound to Atg8 and free Atg7. Atg7 is shown with a ribbon model. Coloring is as in (C).
See also Figure S3.
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6. Figure 4 In Vitro and In Vivo Mutational Analyses
(A) In vitro analyses of the mutational effects on the formation of Atg7∼Atg8 intermediates. Atg7∼Atg8 formation was calculated by dividing the amount of Atg7∼Atg8 intermediate with that of free Atg7. The values and the error bars in (A), (B), and (D) are the means and the standard deviations of three independent experiments, respectively.
(B) In vitro pull-down assay between Atg7 mutants and wild-type Atg8 (left) and between wild-type Atg7 and Atg8 mutants (right). Atg7 binding was calculated by dividing the ratio of eluted Atg7 mutants and GST-Atg8 (left) or eluted Atg7 and GST-Atg8 mutants (right) with that of the wild-type pair.
(C) Immunoblotting analysis of the accumulation level of Atg8-PE in atg7Δ cells expressing Atg7 mutants. Although the band of Atg7ΔC13 was weak, it appears not to be due to the low expression level but to the weak recognition of Atg7ΔC13 by anti-Atg7 antibody as shown in Figure S4.
(D) Autophagic activity in atg7Δ cells expressing Atg7 mutants.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 NMR Structure of the Atg7C30-Atg8 Complex
(A) NMR structure of the Atg8-Atg7C30 complex. Atg8 is shown with surface and ribbon models, while Atg7C30 is shown with a ribbon model on which side chains are shown with stick models. The residues constituting the induced pocket, the W site and the L site of Atg8 are colored orange, red, and green, respectively. Basic residues of Atg8 that surround Atg7C30 are colored blue. The right figure is obtained by 80° rotation of the left figure around the horizontal axis.
(B) In vitro pulldown assay between Atg8 mutants and the wild-type Atg7 (left) and between wild-type Atg8 and Atg7 mutants (right). Atg7 binding was calculated by dividing the ratio of eluted Atg7 and GST-Atg8 mutants (left) or eluted Atg7 mutants and GST-Atg8 (right) with that of the wild-type pair. The values and the error bars are the means and the standard deviations of three independent experiments, respectively.
See also Figure S5.
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8. Figure 6 Mechanism of Atg8 Transfer to Atg3 Catalyzed by Atg7
(A) Structural comparison between Atg7 and NEDD8 E1. The left figure shows the structure of full-length Atg7-Atg8 complex modeled by just superimposing the crystal structure of the Atg7CTD-Atg8 complex onto that of Atg7(1–613). Atg7 and Atg8 are colored as in Figures 1B and 3A for one complex, while for another complex, Atg7 and Atg8 are colored gray and black, respectively. The basic residues crucial for Atg3 binding were colored blue and circled with a broken line. The right figure shows the crystal structure of NEDD8 E1 (Uba3-APPBP1 heterodimer) in complex with Ubc12 and two NEDD8 molecules (PDB ID 2NVU) (Huang et al., 2007). AD, UFD and catalytic cysteine half domain with connecting loops of Uba3 are colored green, cyan and blue, respectively, while APPBP1, Ubc12, NEDD8 bound to the adenylation site [NEDD8(A)], and NEDD8 thioester-linked to NEDD8 E1 [NEDD8(T)] are colored gray, orange, salmon pink and red, respectively. Cys507 of Atg7 and Cys216 of Uba3 are shown with a space-filling model.
(B) In vitro experiments showing that trans reaction is efficient for Atg8 transthiolation. Hetero- and homodimers, as well as mixture of homodimers preincubated for the indicated hours, were incubated with Atg3, Atg8, and MgATP, and the formation level of Atg3∼Atg8 intermediates was analyzed by SDS-PAGE. Asterisks in (B) and (C) indicate nonspecific bands.
(C) In vitro experiments showing that cis reaction is less favored for Atg8 transthiolation. Left: Ribbon representation of the homodimer structure of Atg7(1–613), in which Arg511 and Glu524, which form a salt bridge between each other, are shown with stick models. One Atg7 molecule is colored green, while the other is colored gray. Right: Stabilized heterodimers were incubated with Atg3, Atg8, and MgATP, and the formation level of Atg3∼Atg8 intermediates was analyzed by SDS-PAGE.
See also Figure S6.
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9. Figure 7
Proposed Models of Atg8 Activation and Transfer to Atg3 by Atg7
(A) Activation model. Atg8 (red oval) is initially captured by the C-terminal tail of ECTD (yellow line), and is then transferred to AD (green square) and the C-terminal tail of Atg8 (red line) binds to the catalytic site of Atg7, where Atg8 Gly116 is adenylated and is then thioester-linked to the catalytic cysteine (blue circled C). Upon Atg8 binding to AD, α17 is induced and CL undergoes a large conformational change, both of which recognize Atg8. In this figure, Atg7 is presented as a monomer for simplicity.
(B) Transfer model. Atg8 thioester-linked to the catalytic cysteine of one AD is transferred to the catalytic cysteine of Atg3 bound to the NTD (cyan square) of distinct monomer within a dimer via a trans mechanism. Coloring is as in (A).
See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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