(A) The simulation systems. The NompC was divided into two subsystems, denoted by the cyan and red rectangular boxes, for the molecular dynamics (MD) simulations. (B−D) The transmembrane (TM) pore …
The ion density maps were obtained from the simulation trajectories with a −300 mV transmembrane potential for Na+ ions and a 300 mV transmembrane potential for K+ ions, calculated from the …
(A) Principal component analysis (PCA) of the molecular dynamics (MD) simulation trajectories (FI0, SI0, and CI0 in Supplementary file 1a). The projections on the second eigenvector can distinguish …
The side and bottom views of the S6 and TRP domains before and after pulling or pushing.
(A) The simulation system in which the linker helix (LH) domain (orange) was restrained and a pushing or pulling force was applied to the first AR (gray). (B) Projection of the reaction forces of …
(A) The compression of the ankyrin repeat (AR) region will generate a pushing force and a torque on the linker helix (LH) domain, pointing to the extracellular side. (B) The LH domain further pushes …
(A) The bottom view and (B) the side view of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) molecules moving around H1423. The transient receptor potential (TRP) domain is shown in blue, …
This video shows the TM pore size evolution during the 250 ns ‘free’, ‘pulling’, and ‘pushing’ simulations.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (D. mel) | Schneider 2 (S2) cells | CCTCC (China Center for Type Culture Collection) | Serial# GDC0138 | Cell species report and Mycoplasma contamination test reports provided |
Antibody | Rabbit anti-αNOMPC-EC (polyclonoal) | Ref. (Zhang et al., 2015) | Immunostaining dilution (1: 500), primary antibody | |
Antibody | Alexa Fluor 594 AffiniPure Donkey Anti Rabbit IgG(H + L) | Yeason | Cat# 34212ES60 | Immunostaining dilution (1: 100), secondary antibody |
Recombinant DNA reagent | pActin-Gal4 (plasmid) | Ref. (Yan et al., 2013) | Plasmid for driving Gal4 expression under actin promoter in S2 cells | |
Recombinant DNA reagent | pUAST-NOMPC-EGFP (plasmid) | Ref. (Yan et al., 2013) | Plamid for Gal4-driven NompC expression in S2 cells | |
Recombinant DNA reagent | pUAST-NOMPC-EGFP (del-miniwhite,dm) (plasmid) | This paper | Plamid for Gal4-driven WT NompC expression in S2 cells, no miniwhite sequence | |
Recombinant DNA reagent | pUAST-NOMPC(D1236A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on D1236 | |
Recombinant DNA reagent | pUAST-NOMPC(R1581A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on R1581 | |
Recombinant DNA reagent | pUAST-NOMPC(K1244A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on K1244 | |
Recombinant DNA reagent | pUAST-NOMPC(E1571A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on E1571 | |
Recombinant DNA reagent | pUAST-NOMPC(Q1253A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on Q1253 | |
Recombinant DNA reagent | pUAST-NOMPC(S1577A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on S1577 | |
Recombinant DNA reagent | pUAST-NOMPC(S1421A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on S1421 | |
Recombinant DNA reagent | pUAST-NOMPC(W1572A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on W1572 | |
Recombinant DNA reagent | pUAST-NOMPC(W1115A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on W1115 | |
Recombinant DNA reagent | pUAST-NOMPC(D1142A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on D1142 | |
Recombinant DNA reagent | pUAST-NOMPC(R1127A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on R1127 | |
Recombinant DNA reagent | pUAST-NOMPC(E1163A)-EGFP(dm) (plasmid) | This paper | Contains Drosophila NOMPC CDS with alanine substitution on E1163 | |
Chemical compound, drug | 1-Oleoyl-2-acetyl-sn-glycerol (OAG) | Sigma-Aldrich | Cat# O6754 | DAG analogue |
Chemical compound, drug | GdCl3 | Sigma-Aldrich | Cat# 439770 | NOMPC blocker |
Chemical compound, drug | Concanavalin A (Con A) | Sigma-Aldrich | Cat# C5275 | Cell adhesion |
Chemical compound, drug | ClonExpress II One-step Cloning Kit | Vazyme | Serial# C112 | Site-directed mutagenesis |
Chemical compound, drug | TransIT-Insect Transfection Reagent | Mirus | Cat# MIR 6100 | S2 cell transfection |
Tables.
(a) Molecular dynamics/steered molecular dynamics (MD/SMD) trajectories of system I. (b) MD/SMD trajectories of system II. (c) Ion permeation simulations of the partially opened NompC. (d) Stable hydrogen bonds and their occupancies in the MD/SMD trajectories. (e) Different domains/components used in the MD simulations. (f) The primers used for the alanine substitution.