Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β accumulates at sites of DNA damage in an ATM/H2AX/MDC1-dependent manner. ( A ) U2OS cells were microirradiated, fixed 5 min later, and immunostained for WRAP53β and γH2AX, a marker for DNA DSBs. Nuclei were stained with DAPI in all immunofluorescence experiments. ( B ) U2OS cells were treated with siControl or two different WRAP53β targeting oligonucleotides (siWRAP53#1 and siWRAP53#2) for 48 h, irradiated (6 Gy, 1-h recovery) or left untreated, fixed after pre-extraction with cytoskeleton (CSK) buffer, and immunostained for WRAP53β and γH2AX. ( C ) U2OS cells were irradiated (6 Gy), fixed, and immunostained for WRAP53β at the indicated time points. Quantification is given as the percentage of the 100 cells counted in each experiment whose nuclei contained WRAP53β IRIF. ( D ) U2OS cells were treated with the inhibitors or siRNAs, as indicated, for 6 h or 48 h, respectively; irradiated (6 Gy, 1-h recovery); fixed after pre-extraction with CSK buffer; and immunostained for WRAP53β and γH2AX. ( E ) Quantification of the results in D , as the percentage of the 100 cells counted in each experiment whose nuclei contained WRAP53β IRIF. The error bars depict the SEM; n = 3; (***) P < 0.001 as determined by Student’s t -test. ( F ) ChIP assay showing the recruitment of WRAP53β to the I-PpoI-induced DSB at chromosome 1 in MCF7 cells stably expressing ddI-PpoI. The time indicated is hours after the addition of 4-OHT. The I-PpoI cleavage site on chromosome 1 is located at distance 0. Cells were cultivated in medium containing 0.1% FBS for 24 h before DSB induction. Data are shown as the mean of two independent experiments. The Y -axis displays the fold change in relative occupancy normalized to the control.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Marker, Staining, Immunofluorescence, Irradiation, Extraction, Stable Transfection, Expressing, Control

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β promotes recruitment of repair proteins to DSBs. ( A ) U2OS cells were transfected with siControl or siWRAP53#2 oligonucleotides for 24 h, exposed to IR (6 Gy) or left untreated, and, 1 h later, immunostained for γH2AX, MDC1, BRCA1, 53BP1, and RAD51. ( B ) U2OS cells treated as in A and then immunostained for RNF168 and conjugated ubiquitin (with the FK2 antibody). In the case of GFP-RNF8 staining, following treatment with oligonucleotides for 24 h, the cells were transiently transfected with the GFP-RNF8 plasmid for 8 h, exposed to IR (6 Gy), allowed to recover for 1 h, and then fixed and analyzed. ( C ) Quantification of the results in A and B as the percentage of 200 cells counted in each experiment whose nuclei contained IRIF. In the case of GFP-RNF8, only successfully transfected cells were counted. ( D ) U2OS cells were treated with the siRNAs indicated for 24 h, irradiated (6 Gy), allowed to recover for 1 h, and then subjected to Western blotting for WRAP53β, H2AX, and β-actin. The error bars depict the SEM. n = 3; (**) P < 0.01; (***) P < 0.001, as determined by Student’s t -test.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Transfection, Ubiquitin Proteomics, Staining, Plasmid Preparation, Irradiation, Western Blot

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β binds MDC1 and RNF8 via their FHA domains. ( A ) U2OS cells were either left untreated or irradiated with 6 Gy of IR, and, 30 min, later immunoprecipitation of WRAP53β was performed, followed by immunoblotting of WRAP53β, MDC1, GFP-RNF8, and β-actin. ( B ) U2OS cells were transfected with the indicated HA-MDC1 constructs for 16 h and irradiated with 2 Gy, and, 30 min later, immunoprecipitation of WRAP53β was performed, followed by immunoblotting of WRAP53β and HA-MDC1. ( C ) Schematic illustration of RNF8 deletion constructs. ( D ) U2OS cells were transiently transfected with EGFP-RNF8 plasmids, HA-MDC1, and Flag-WRAP53β for 16 h; irradiated; and subjected to immunoprecipitation of GFP followed by immunoblotting for GFP-RNF8, Flag-WRAP53β, and HA-MDC1. (HC) Heavy chain of the antibody. U2OS ( E ) and H1299 ( F ) cells were transiently transfected with Flag-RNF8 plasmids, HA-MDC1, and EGFP-WRAP53β for 16 h; irradiated; and subjected to Flag immunoprecipitation followed by immunoblotting for the indicated proteins. ( G ) Schematic illustration of the domain architecture of MDC1 and RNF8, where black lines mark WRAP53β- and MDC1-binding sites. Numbers indicate amino acids.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Irradiation, Immunoprecipitation, Western Blot, Transfection, Construct, Binding Assay

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β facilitates MDC1–RNF8 interaction through its WD40 domain. ( A ) U2OS cells were treated with the siRNAs indicated for 48 h and with GFP-RNF8 for 24 h (all samples), irradiated with 6 Gy, and, 30-min later, subjected to immunoprecipitation of WRAP53β followed by immunoblotting of WRAP53β, MDC1, RNF8, γH2AX, and β-actin. ( B ) Immunoprecipitation of MDC1 in irradiated (6 Gy, 15-min recovery) U2OS cells treated with the siRNA indicated for 48 h or ATM inhibitor (ATMi) for 24 h. All samples were transfected with GFP-RNF8 for 16 h. ( C ) U2OS cells were treated with the siRNAs indicated for 48 h or ATM inhibitor for 16 h, irradiated with 6 Gy, allowed to recover for 15 min, and then subjected to Western blotting of MDC1, WRAP53β, γH2AX, and β-actin. ( D ) Schematic illustration of EGFP-tagged deletion constructs of WRAP53β. ( E ) U2OS cells were transiently transfected with the indicated EGFP-WRAP53β plasmids and Flag-RNF8 for 16 h, irradiated, and subjected to GFP immunoprecipitation followed by immunoblotting for MDC1, Flag-RNF8, and GFP-WRAP53β. (HC) Heavy chain of the antibody. ( F ) U2OS cells were transfected with siControl or siWRAP53#2 oligonucleotides for 8 h followed by transfection of EGFP-Empty or EGFP-WRAP53β WD40 (1–7) for 16 h, exposed to IR (6 Gy), and, after 1 h, immunostained for 53BP1 followed by quantification of the results. The graph in A shows the percentage of 100 GFP transfected cells in each experiment whose nuclei were 53BP1-positive. The error bars depict the SEM. n = 3; (*) P < 0.05, as determined by Student’s t -test. ( G ) U2OS cells were transiently transfected with the indicated EGFP-WRAP53β plasmids, HA-MDC1, and Flag-RNF8 for 16 h; irradiated; and subjected to immunoprecipitation of GFP followed by immunoblotting for HA-MDC1, Flag-RNF8, and GFP-WRAP53β. ( H ) Schematic illustration of how WRAP53β scaffolds the MDC1–RNF8 complex. Upon DNA damage, WRAP53β binds MDC1 and RNF8 simultaneously via its WD40 domain and facilitates their interaction.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Irradiation, Immunoprecipitation, Western Blot, Transfection, Construct

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β promotes HR and NHEJ. ( A ) U2OS cells were treated with the siRNAs indicated for 24 h, exposed to 6 Gy of IR, fixed 1 h or 24 h later, and immunostained for γH2AX. ( B ) Quantification of the results in A showing the percentage of nuclei containing >10 γH2AX foci ( n = 200). ( C , D ) HR ( C ) and NHEJ ( D ) efficiency following treatment of the cells with the siRNA indicated for 48 h. DR-GFP (HR) and EJ5-GFP (NHEJ) reporter systems were used in the FACS analysis, with siRAD51 and siArtemis as positive controls. ( E ) Cells were transfected with siRNA for 24 h, exposed to IR (3 Gy), harvested at the time points indicated, and subjected to flow cytometry. Nonirradiated cells were treated with siRNA alone for 60 h. The error bars depict the SEM. n = 3; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, as determined by Student’s t -test.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Transfection, Flow Cytometry

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β protects cells against accumulation of spontaneous DNA damage. ( A ) U2OS cells were treated with siControl or two different siWRAP53 oligonucleotides (siWRAP53#1 and siWRAP53#2) for 24 h, fixed, and immunostained for WRAP53β and γH2AX. ( B ) The percentage of nuclei in A containing >10 γH2AX foci was quantified in the 200 cells counted for each experiment. ( C ) After treating U2OS with siWRAP53#2 or siControl for 24 h or 48 h, DNA damage was assessed by the alkaline comet assay. The error bars depict the SEM. n = 3; (**) P < 0.01; (***) P < 0.001, as determined by Student’s t -test.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Alkaline Single Cell Gel Electrophoresis

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: Schematic model of WRAP53β function in the DDR pathway. (Step 1) In response to IR, γH2AX and MDC1 accumulate at DSBs independently of WRAP53β. ATM-mediated phosphorylation of MDC1 makes MDC1 competent to bind RNF8. However, RNF8 is not yet localized at DSBs. (Step 2) WRAP53β is recruited to sites of DNA damage in an ATM-, H2AX-, and MDC1-dependent manner. Simultaneous binding of MDC1 and RNF8 to WRAP53β facilitates their direct interaction and retention of RNF8 at DSBs. (Step 3) Once assembled at DSBs, RNF8 catalyzes ubiquitylation of H2AX. Ubiquitylation at DSBs enables recruitment and accumulation of 53BP1, BRCA1, and RAD51 and subsequent DNA repair.

Article Snippet: The WRAP53β antibodies used were rabbit α-WRAP53-C2 (used for Western blot, immunoprecipitation, ChIP, and immunofluorescence experiments; Innovagen AB, catalog no. PA-2020-100), rabbit α-WDR79 (used for immunofluorescence; Bethyl Laboratories, catalog no. A301-442A-1), rabbit α-WRAP53 (used for immunofluorescence; Proteintech, catalog no. 14761-1-AP), rabbit α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-D01P), mouse monoclonal α-WDR79 (clone 1F12; used for immunofluorescence; Abnova, catalog no. H00055135-M04), and mouse polyclonal α-WDR79 (used for immunofluorescence; Abnova, catalog no. H00055135-B01P).

Techniques: Phospho-proteomics, Binding Assay

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Representative intracellular calcium signal of AD293 cells transfected with hTRPC5 (1-764) evoked by 256 nM EA ( A ) or 14 mM extracellular calcium ( B, C ), and inhibited by different concentrations of HC-070 ( B ) or clemizole (CMZ) ( C ), measured by FLIPR calcium assay. Arrow 1 denotes the time point for application of buffer alone or buffer with inhibitors; arrow 2 denotes the time point for application of the activators. ( D ) Fluorescence size exclusion chromatography profile of full-length hTRPC5 and hTRPC5 (1-764) . ( E ) Representative size exclusion chromatography of hTRPC5 (1-764) purified in glycol-diosgenin (GDN) micelles. ( F ) SDS-PAGE gel of hTRPC5 (1-764) eluted from size exclusion chromatography. The position of TRPC5 is labeled. Fractions that were pooled for cryo-EM analysis are denoted by asterisks.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Transfection, Calcium Assay, Fluorescence, Size-exclusion Chromatography, Purification, SDS Page, Labeling, Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A, B ) Inhibitory effect of clemizole (CMZ) ( A ) and HC-070 ( B ) on the extracellular calcium-induced intracellular calcium increase of cells with wild-type hTRPC5 over-expression measured by FLIPR calcium assay (data are shown as means ± standard error, n = 3 independent experiments). ( C, D ) Cryo-EM density maps of CMZ-bound hTRPC5 shown in side view ( C ) and top view ( D ). Subunits A, B, C, and D are colored in purple, light blue, orange, and gray, respectively. Lipids are colored in gold. The approximate boundary of the cell membrane is indicated by gray lines. TMD, transmembrane domain; ICD, intracellular cytosolic domain. ( E ) Ribbon diagram of a single subunit with secondary structure elements represented in different colors. ARD, ankyrin repeats domain; LHD, linker-helix domain; CH1, C-terminal helix 1; CH2, C-terminal helix 2. Figure 1—source data 1. Inhibition of WT hTRPC5 by clemizole or by HC-070.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Over Expression, Calcium Assay, Cryo-EM Sample Prep, Inhibition

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A ) Representative raw micrograph recorded on K2 Summit camera. ( B ) Representative 2D class averages of apo hTRPC5. ( C ) Flowchart for cryo-EM data processing of apo hTRPC5. ( D ) Fourier shell correlation (FSC) curves of the two independently refined maps for unmasked (blue line, 3.9 Å) and corrected (purple line, 3 Å). Resolution estimation was based on the criterion of FSC 0.143 cut-off. ( E ) Angular distribution of clemizole (CMZ)-bound hTRPC5. This is a standard output from cryoSPARC. ( F ) FSC curve of the refined model versus EM map.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Cryo-EM map of apo hTRPC5 colored by local resolution, shown in top view ( A ), side view ( B ), and cross-section ( C ). The position of cross-section is shown as a dashed line in ( A ). ( D ) Cryo-EM density map (contoured at 4.1 σ, gray mesh) with atomic models superimposed.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A ) Representative raw micrograph recorded on K2 Summit camera. ( B ) Representative 2D class averages of CMZ-bound hTRPC5. ( C ) Flowchart for cryo-EM data processing of CMZ-bound hTRPC5. ( D ) Fourier shell correlation (FSC) curves of the two independently refined maps for unmasked (blue line, 3.5 Å) and corrected (purple line, 2.7 Å). Resolution estimation was based on the criterion of FSC 0.143 cut-off. ( E ) Angular distribution of CMZ-bound hTRPC5. This is a standard output from cryoSPARC. ( F ) FSC curve of the refined model versus EM map.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Cryo-EM map of HC-070-bound hTRPC5 colored by local resolution, shown in top view ( A ), side view ( B ), and cross-section ( C ). The position of cross-section is shown as dashed line in ( A ). ( D ) Cryo-EM density map (contoured at 3 σ, gray mesh) with atomic model superimposed. ( E–F ) Cryo-EM density map of HC-070 shown in different views contoured at 3 and 7 σ, respectively. ( G–J ) Cryo-EM densities of HC-070-binding pocket in ( G ) HC-070-bound, ( H ) clemizole (CMZ)-bound, ( I ) apo, and ( J ) Pico145-bound hTRPC5 maps, contoured at 3.8 σ.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A–C ) Cryo-EM map of CMZ-bound hTRPC5 colored by local resolution, shown in top view ( A ), side view ( B ), and cross-section ( C ). The position of cross-section is shown as a dashed line in ( A ). ( D ) Cryo-EM density map (contoured at 3.8 σ, gray mesh) with atomic models superimposed. ( E ) Cryo-EM density of CMZ shown in different views (contoured at 3.7 σ). ( F ) Cryo-EM densities of CMZ-binding pocket in CMZ-bound hTRPC5 map (contoured at 4.6 σ). ( G, H ) Cryo-EM densities in HC-070-bound hTRPC5 and apo hTRPC5 maps corresponding to the CMZ-binding pocket contoured at the same σ as in ( F ).

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: eLife

Article Title: Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

doi: 10.7554/eLife.63429

Figure Lengend Snippet: ( A ) Comparison of experimental density map of diacylglycerol (DAG) (gray) (contoured at 3.2 σ) and simulated density map of lysophospholipid (yellow). Please note, the density for electron-dense phosphate head group is absent in the cryo-EM maps obtained experimentally. ( B–D ) Close-up views of lipid 1 ( B ), lipid 2 ( C ), and cholesteryl hemisuccinate (CHS) ( D ) binding sites. Subunits A and B are colored in purple and light blue, respectively. Residues that interact with lipids are shown as sticks. Insets show cryo-EM densities of these lipids, contoured at 2.3, 3.1, and 3.1 σ, respectively.

Article Snippet: The following datasets were generated: Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole Electron Microscopy Data Bank EMD-30575 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with Clemizole RCSB Protein Data Bank 7D4P Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 Electron Microscopy Data Bank EMD-30576 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in complex with HC-070 RCSB Protein Data Bank 7D4Q Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state Electron Microscopy Data Bank EMD-30987 Wei M Song K Chen L 2020 cryo-EM structure of hTRPC5 in apo state RCSB Protein Data Bank 7E4T The following previously published dataset was used: Tang Q Guo W Zheng L Wu JX Liu M Zhou X Zhang X Chen L 2018 Cryo-EM structure of human TRPC6 at 3.8A resolution Electron Microscopy Data Bank EMD-6856

Techniques: Cryo-EM Sample Prep, Binding Assay