lipids 840035 dioleoyl phosphatidylinositol dopi avanti polar lipids 850149 phosphatidic acid  (Croda International Plc)

Journal: bioRxiv

Article Title: Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane

doi: 10.1101/2022.07.26.501514

Figure Lengend Snippet: (A) Atg18-PR72AA refined atomic model with surface colored by electrostatics, FRRG motif in pink and phosphate binding sites from aligned PDB-ID 5LTD. (B) Diamond shaped tetramer unit (green) with FRRG motifs (red) and their adjacent phosphate binding sites, which are buried in Atg18 tubes. (C) Pelletation assay (top) and negative stain of pellet fractions from Atg18-WT (left) and Atg18-FGGG (right) (D) Top. Chemical formulas of soluble PIP derivates diC8-PI3P, diC8-PI(3,5)P 2 and diC8-PI(4,5)P 2 used in binding experiments. Bottom. Negative stain electron micrographs of Atg18 nontreated or treated with 4x molar excess of the indicated PIP derivate preventing tube formation. (E) Pelletation assay with corresponding SDS-PAGE of the supernatant (SN) and the pellet (Pe) fraction and the negative stain EM micrographs of two pellets. (F) Tubes from the negative control were treated with PIP derivates. Only PI(3,5)P 2 resulted in tube disassembly.

Article Snippet: LUVs were prepared with a lipid content of 95% DOPC and 5% DO-PI(3,5)P 2 (Avanti Polar Lipids).

Techniques: Binding Assay, Staining, SDS Page, Negative Control

Journal: bioRxiv

Article Title: Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane

doi: 10.1101/2022.07.26.501514

Figure Lengend Snippet: (A) Gel filtration profile of Atg18-WT (black) and Atg18-FGGG (rose). Uncropped and unedited Coomassie-stained SDS-PAGE gels of pelletation assay used for preparing : (B) FGGG mutant (left) in comparison with Atg18-WT (left) and (C) Atg18 incubated with water (control), soluble DiC8-PI3P, DiC8-PI(3,5)P 2 and DiC8-PI(4,5)P 2 as indicated on top of the lanes.

Article Snippet: LUVs were prepared with a lipid content of 95% DOPC and 5% DO-PI(3,5)P 2 (Avanti Polar Lipids).

Techniques: Filtration, Staining, SDS Page, Mutagenesis, Incubation

Journal: bioRxiv

Article Title: Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane

doi: 10.1101/2022.07.26.501514

Figure Lengend Snippet: (A) Volume density rendering of a reconstructed tomogram containing Atg18-WT and PI(3,5)P 2 -containing large unilamellar vesicles (LUVs). (B) Central slice of the corresponding tomogram acquired with Volta phase plate (averaged five slices to display increased contrast). (C) Examples of rotational 2D classification after subvolume extraction of double-membrane subtomograms. The 60 Å thick membrane bilayers are spaced apart by 80 Å. (D) Initial subvolume average dominated by membrane density contributions. (E) Focused 3D classes after membrane subtraction reveal protein densities. (F) Refined structure of the selected 3D class (C1 symmetry) shows organized stacking of eight disc-shaped molecules. (G) Density fit with four Atg18-WT dimers formed by I-interface previously observed in Atg18 tubular assemblies and soluble dimers. The Atg18 molecule within the dimer is colored orange and magenta, respectively. The remaining six Atg18 molecules are colored grey. FRRG motif is highlighted in red and allows for direct binding of opposing membranes. (H) Slice through reconstructed tomogram reveals Atg18 dimer stacking with the approx. 45° angle relative to the membrane bilayer planes.

Article Snippet: LUVs were prepared with a lipid content of 95% DOPC and 5% DO-PI(3,5)P 2 (Avanti Polar Lipids).

Techniques: Binding Assay

Journal: Structure (London, England : 1993)

Article Title: Ca 2+ -dependent lipid preferences shape synaptotagmin-1 C2A and C2B dynamics: Insights from experiments and simulations.

doi: 10.1016/j.str.2024.07.017

Figure Lengend Snippet: Figure 2. C2A and C2B domains bind negatively charged phospholipids (A) Lipid overlay assays. Membrane lipid strips spotted with different membrane lipids were incubated with C2A (lhs, cyan) and C2B (rhs, purple). Protein binding was visualized using specific antibodies. (B) Native mass spectrometry reveals binding of C2A (lhs, cyan) and C2B (rhs, purple) to up to three DOPS (red) and DOPI(4,5)P2 (yellow) molecules. See also Figures S1 and S2.

Article Snippet: St. Louis, U.S.A. cat. no. A9044; RRID: AB_258431 Bacterial and virus strains E. coli BL21 (DE3) New England Biolabs cat. no. C2527I Chemicals, peptides, and recombinant proteins DOPS Avanti Polar Lipids cat. no. 840035 DOPC Avanti Polar Lipids cat. no. 850375 DOPE Avanti Polar Lipids cat. no. 850725 DOPI Avanti Polar Lipids cat. no. 850149 DOPI(4)P Avanti Polar Lipids cat. no. 850151 DOPI(4,5)P2 Avanti Polar Lipids cat. no. 850155 DOPI(3,4,5)P3 Avanti Polar Lipids cat. no. 850156 Cholesterol Avanti Polar Lipids cat. no. 700100 DAG Avanti Polar Lipids cat. no. 800815 Critical commercial assays Membrane lipid strips Echelon Biosciences cat. no. P-6002 Deposited data MD trajectory files This paper https://doi.org/10.5281/zenodo.10478556 Python scripts for MD analysis This paper https://github.com/cschmidtlab/ syt1_membrane_md Recombinant DNA pET28a Syt-1 C2A Rattus Norvegicus Syt-1 (97-263) Stein et al.60 N/A pET28a Syt-1 C2B Rattus Norvegicus Syt-1 (271-421) Stein et al.60 N/A Software and algorithms SpectraManager JASCO N/A MassLynx v4.1 Waters N/A Kalliope Anton Paar N/A Gromacs 2020.4 Berendsen et al.61 www.gromacs.org CHARRM-Gui Jo et al.62; Lee et al.63 www.charmm-gui.org vmd Humphrey et al.64 https://www.ks.uiuc.edu/Research/vmd/ md-python v3.0.6 www.python.org N/A SESCA Nagy et al.65 https://www.mpinat.mpg.de/sesca Unidec Marty et al.66 https://github.com/michaelmarty/ UniDec/releases MDanalysis v2.5.0 Michaud-Agrawal et al.67 www.mdanalysis.org open source PyMol www.pymol.org https://github.com/schrodinger/ pymol-open-source Structure 32, 1–14.e1–e5, October 3, 2024 e1

Techniques: Membrane, Incubation, Protein Binding, Mass Spectrometry, Binding Assay