prk7  (Addgene inc)

Journal: Science Advances

Article Title: Structure of the human TSC:WIPI3 lysosomal recruitment complex

doi: 10.1126/sciadv.adr5807

Figure Lengend Snippet: ( A ) Focused view of the TSC HR dimer. A single subunit of the TSC1 HR dimer mediates contacts to the TSC1 CC domain and the TSC2 HRs. Two regions of the large extended loop (TSC1 IDR1) shield the exposed second binding site of the TSC1 N-terminal dimer. Top: Schematic of the TSC1 HR dimer in context of TSC. ( B ) The TSC1 HR dimer clamps onto a hydrophobic helix of the TSC1 CC domain. The second TSC1 helix is hidden for clarity [shown in (A), dark blue]. ( C ) Key contacts in the TSC1 HR dimer interface mediated by the first TSC1 extended loop (IDR1). ( D ) Focused view of the WIPI3:TSC1 interaction as resolved by cryo-EM. ( E ) The 3.17-Å crystal structure of WIPI3 in complex with the TSC1 WIR motif. Bottom: Schematic of the WIPI3 binding site in context of TSC.

Article Snippet: TBC1D7 was polymerase chain reaction amplified from pET-28a TBC1D7 (Addgene plasmid no. 32047 was a gift from C. Arrowsmith) and cloned into pcDNA 3.1 (Thermo Fisher Scientific) with a C-terminal FLAG-tag. pRK7 FLAG TSC2 (Addgene plasmid no. 8996) was a gift from J. Blenis, and pcDNA 3.1 myc TSC1 (Addgene plasmid no. 12133) was a gift from C. Walker.

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Science Advances

Article Title: Structure of the human TSC:WIPI3 lysosomal recruitment complex

doi: 10.1126/sciadv.adr5807

Figure Lengend Snippet: ( A ) Cα atoms of all disease-associated missense mutations are rendered as spheres of size and color proportional to the frequency of observation (COSMIC, LOVD, and HGMD) ( – ). Disease-associated mutations cluster to the TSC1 HR PIP-binding dimer (left), as well as the TSC2 Rap-GAP central core (right). ( B ) Surface rendering of the complete human TSC with RHEB modelled according to AlphaFold . The binding sites of RHEB, TSC1:PI(3)P, and WIPI3:PI(3)P define a membrane binding plane that is consistent with a singly occupied RHEB-binding model of the TSC:WIPI3:RHEB lysosomal mTORC1 inhibitory complex.

Article Snippet: TBC1D7 was polymerase chain reaction amplified from pET-28a TBC1D7 (Addgene plasmid no. 32047 was a gift from C. Arrowsmith) and cloned into pcDNA 3.1 (Thermo Fisher Scientific) with a C-terminal FLAG-tag. pRK7 FLAG TSC2 (Addgene plasmid no. 8996) was a gift from J. Blenis, and pcDNA 3.1 myc TSC1 (Addgene plasmid no. 12133) was a gift from C. Walker.

Techniques: Binding Assay, Membrane