Journal: Science Advances

Article Title: Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

doi: 10.1126/sciadv.adg2997

Figure Lengend Snippet: ( A ) Domain schematic of autophagy-related protein 9A (ATG9A), ATG13, and ATG101. HORMA, Hop1/Rev7/Mad2; TMD, transmembrane domain. ( B ) MBP pull-down assay using recombinant MBP-ATG9A in DDM/CHS micelles, MBP-FOLDON-ATG9A, and MBP-ATG9A constructs bound to amylose resin. Incubated with ATG13 HORMA /TwinStrep-Flag (TSF)–ATG101 and visualized on 4 to 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel. ( C ) Isothermal titration calorimetry (ITC) of MBP-ATG9A (801–839) and ATG13 HORMA :TSF-ATG101. DP, differential power. ( D ) Glutathione S -transferase (GST) pull-down assay using purified TSF-ATG13 HORMA :GST-ATG101 incubated with lysate containing ATG9A wild type (WT) or mutants. Bound protein was run on 4 to 12% SDS-PAGE gel. ( E ) Quantification of GST pull-down assays from (D) showing SD ( n = 3). Two-sample t test was used to compare wild-type to mutant pull-down results. P values are as follows: * P < 0.1; ** P < 0.05; NS, not significant.

Article Snippet: The fusion construct of human ATG9A C terminus with ATG101 for crystallization was subcloned as an N-terminal GST tag, Tobacco etch virus (TEV) cleavage site, ATG9 (828–839), and 5–amino acid linker (GSDEA), followed by ATG101 (1–198) into the pCAG-eGFP vector [Research Resource Identifier (RRID): Addgene_89684].

Techniques: Pull Down Assay, Recombinant, Construct, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Isothermal Titration Calorimetry, Purification, Mutagenesis

Journal: Science Advances

Article Title: Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

doi: 10.1126/sciadv.adg2997

Figure Lengend Snippet: ( A ) Multisequence alignment of ATG9A C-terminal tails performed using ClustalO and aligned to ATG9A from Hs. Hs, Homo sapiens ; Sp, Schizosaccharomyces pombe ; Bt, Bos taurus ; Pa, Pongo abelii ; Mm, Mus musculus ; Dr, Danio rerio ; Ce, Caenorhabditis elegans ; At, Arabidopsis thaliana ; Dm, Drosophila melanogaster . ( B ) Constructs used for crystallization including ATG13 HORMA (1–197) and fused ATG9A (829–839)–GlySerLinker-ATG101 (1–198). ( C ) Ribbon representation of the ATG9A HORMA dimer–interacting region (HDIR)–ATG101 and ATG13 HORMA constructs for crystallography colored by protein. ATG13 HORMA , light blue; ATG101, light green; ATG9A, purple. ( D ) Omit map of the ATG9A HDIR shown at 2.9σ. ( E ) Overlay of the crystal structure and AlphaFold2 (AF2)–predicted model aligned to the ATG13 HORMA domain. Crystal structure colored as in (C); AF2 model colored as follows: ATG13 HORMA , light blue; ATG101, yellow; and ATG9A tail, red. RMSD, root mean square deviation.

Article Snippet: The fusion construct of human ATG9A C terminus with ATG101 for crystallization was subcloned as an N-terminal GST tag, Tobacco etch virus (TEV) cleavage site, ATG9 (828–839), and 5–amino acid linker (GSDEA), followed by ATG101 (1–198) into the pCAG-eGFP vector [Research Resource Identifier (RRID): Addgene_89684].

Techniques: Construct, Crystallization Assay

Journal: Science Advances

Article Title: Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

doi: 10.1126/sciadv.adg2997

Figure Lengend Snippet: ( A ) Close-up of the hydrogen network between ATG13 HORMA and the ATG9A HDIR. ( B ) The extended β sheet between ATG101 and ATG9A. ( C ) View of the hydrophobic groove across the HORMA dimer interface, colored by hydrophobicity. ( D ) GST pull-down assay using purified wild-type TSF-ATG13 HORMA :GST-ATG101 and listed mutants incubated with lysate containing MBP-FOLDON-ATG9A. Position of mutants E83 and WF finger shown in Fig. 2C . ( E ) Quantification of GST pull-down assays from (D) showing SD ( n = 3). Two-sample t test was used to compare wild type to mutant pull-down results. P values are labeled as follows: * P < 0.05; ** P < 0.005.

Article Snippet: The fusion construct of human ATG9A C terminus with ATG101 for crystallization was subcloned as an N-terminal GST tag, Tobacco etch virus (TEV) cleavage site, ATG9 (828–839), and 5–amino acid linker (GSDEA), followed by ATG101 (1–198) into the pCAG-eGFP vector [Research Resource Identifier (RRID): Addgene_89684].

Techniques: Pull Down Assay, Purification, Incubation, Mutagenesis, Labeling

Journal: bioRxiv

Article Title: SunTag-PE: a modular prime editing system enables versatile and efficient genome editing

doi: 10.1101/2023.01.13.524016

Figure Lengend Snippet: a , SunTag-PE consists of GCN4 fused to the N-terminus of nCas9. b , PE-SunTag consists of GCN4 fused to the C-terminus of nCas9. c , Two-plasmid system of SunTag-PE, one plasmid expressing pegRNA and scFv-RT, and the other expressing n×GCN4 tethered nCas9. d , Different configurations of SunTag-PE and PE-SunTag (the copy number of GCN4 was 1, 2, 3, 5, and 10). e , Comparison of CTT insertion efficiencies of different SunTag-PE2 and PE2-SunTag configurations in HEK3 locus by deep sequencing. f , Comparison of CTT insertion efficiencies of SunTag-PE2 with N-1×GCN4, deficient SunTag-PE2, Split-PE2, MS2-PE2, and canonical fused PE2 in HEK3 locus. Data and error bars indicate the mean and s.d. of three independent biological replicates.

Article Snippet: The scFv-RT fragment was constructed by replacing nCas9 with the scFv sequence in PE2 plasmid, and this intermediate vector was subsequently assembled with PCR amplified U6-pegRNA fragment, finally obtained U6-pegRNA-CMV-scFv-RT plasmids. n×GCN4 sequences were derived from plasmid (Addgene #113022) via PCR, then N terminus or C terminus n×GCN4 of nCas9 plasmids were constructed, respectively.

Techniques: Plasmid Preparation, Expressing, Sequencing