ion chromatography  (Thermo Fisher)

Journal: ACS infectious diseases

Article Title: Broad-Spectrum Activity and Mechanisms of Action of SQ109 on a Variety of Fungi.

doi: 10.1021/acsinfecdis.5c00210

Figure Lengend Snippet: Figure 4. Effects of SQ109 on Ca2+ uptake/release in S. cerevisiae (ATCC BJ3505) vacuoles as monitored by Cal520-dextran fluorescence, together with vacuole fusion results. (a) Ca2+ uptake/ release at 3 SQ109 concentrations. (b) Ca2+ uptake at 8 min. Ordinary one-way unpaired ANOVA for multiple comparisons was performed with “ATP” as a control. Error bars are mean ± SD. Dunnett multiple comparison test was used for individual p values (n = 3). *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001. (c) Ca2+ release at 30 min. Ordinary one-way unpaired ANOVA for multiple comparisons was performed with “ATP” as a control. Error bars are mean ± SD. Dunnett multiple comparison test was used for individual p values (n = 3). *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001. (d,a) Ca2+ uptake/release at 8 SQ109 concentrations. SQ109 added at t = 0. (e) Dose response curve for inhibition of Ca2+ uptake. The IC50 value is 35 μM. (f) Effects of SQ109 on vacuole fusion. The IC50 is 29 μM (n = 4).

Article Snippet: Calibration was carried out using buffered Ca2+ standards (Invitrogen).

Techniques: Fluorescence, Control, Comparison, Inhibition

Journal: bioRxiv

Article Title: Cryo-EM structures and functional properties of CALHM channels of the human placenta

doi: 10.1101/2020.02.11.944231

Figure Lengend Snippet: Functional characterization of CALHM channels expressed in X. laevis oocytes. ( A ) Western blot of proteins located in the plasma membrane of X. laevis oocytes heterologously expressing the indicated CALHM channels. Protein was isolated after surface-biotinylation by binding to avidin-resin. The proteins are detected with specific antibodies recognizing the respective CALHM paralog. Left, CALHM2, center CALHM4, right CALHM6. The blot demonstrates the targeting of all three paralogs to the plasma membrane. ( B ) Electrophysiological characterization of X. laevis oocytes heterologously expressing the paralogs CALHM1, CALHM2, CALHM4 and CALHM6 in comparison to control oocytes (neg.) recorded at extracellular solutions either containing 3 mM Ca 2+ (3) or 0.5 mM EDTA (Ca 2+ -free, 0). Data show currents of individual oocytes (circle) recorded by two-electrode voltage-clamp (TEVC) at 60 (light blue) and −60 mV (light red). Averages are shown as bars in red (−60 mV) and blue (60 mV), respectively. Dashed lines indicate mean current levels of control oocytes (neg.) recorded in Ca 2+ -free extracellular solutions at 60 and −60 mV. ( C ), Rectification of steady-currents of oocytes displayed in (B) expressed as I 60mV /I −60mV calculated for individual oocytes at 3 mM Ca 2+ (3) and in Ca 2+ -free solutions (0) and averaged. The large value of CALHM1 reflects the activation of the protein at positive voltage in presence of Ca 2+ . ( D ) Ca 2+ -dependence of activation. Change of steady-state currents of oocytes displayed in (B) after Ca 2+ -removal expressed as I noCa2+ /I Ca2+ calculated from individual oocytes at −60 mV (n) and 60 mV (p) and averaged. The large value of CALHM1 at −60 mV reflects the strong activation of currents at negative voltages upon Ca 2+ -depletion. C, D, The difference between the corresponding values of the CALHM paralogs 2, 4, and 6 and neg. are statistically insignificant (as judged by a Student t-test). B-D, Data show averages of 27 (neg.), 21 (CALHM1), 26 (CALHM2), 19 (CALHM4) and 21 (CALHM6) oocytes respectively. Errors are standard deviations.

Article Snippet: For structure determination of human CALHM2, 4 and 6 in the presence of Ca 2+ and of human CALHM4 in the absence of Ca 2+ by cryo-EM, 2.5 μl samples of GDN-purified proteins at a concentration of 1.5-3 mg ml −1 were applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3 or R0.6/1 Au 200 mesh).

Techniques: Functional Assay, Western Blot, Expressing, Isolation, Binding Assay, Avidin-Biotin Assay, Activation Assay

Journal: bioRxiv

Article Title: Cryo-EM structures and functional properties of CALHM channels of the human placenta

doi: 10.1101/2020.02.11.944231

Figure Lengend Snippet: Electrophysiology traces. ( A ) Representative current traces of CALHM1 recorded at indicated extracellular Ca 2+ concentrations. The voltage protocol is shown as inset (left). ( B-F ) Representative currents of CALHM paralogs ( B ), CALHM1, ( D), CALHM2, ( E ), CALHM4 and ( F ), CALHM6 in comparison to ( C ), control oocytes (neg.). The current protocol is shown left. Traces including a step to −60 mV are colored in red, traces including a step to 60 mV in blue. A-F, Data were recorded 40-60 hours after injection of cRNA.

Article Snippet: For structure determination of human CALHM2, 4 and 6 in the presence of Ca 2+ and of human CALHM4 in the absence of Ca 2+ by cryo-EM, 2.5 μl samples of GDN-purified proteins at a concentration of 1.5-3 mg ml −1 were applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3 or R0.6/1 Au 200 mesh).

Techniques: Injection

Journal: bioRxiv

Article Title: Cryo-EM structures and functional properties of CALHM channels of the human placenta

doi: 10.1101/2020.02.11.944231

Figure Lengend Snippet: Cryo-EM reconstruction of CALHM4 in presence of Ca 2+ . ( A ) Representative cryo-EM micrograph acquired with a Tecnai G 2 Polara microscope. ( B ) 2D class averages of CALHM4 in presence of Ca 2+ . ( C ) Data processing workflow. Two rounds of non-symmetrized 3D classification allowed to isolate two populations representing decameric and undecameric assemblies. The particles were further refined with either D10 or D11 symmetry imposed. After performing per-particle CTF refinement and Bayesian polishing, the remaining structural heterogeneity in decamers was segregated by performing the final 3D classification, where the orientations were kept fixed as in the consensus model. To further improve the resolution of each reconstruction, partial signal subtraction followed by 3D refinement was applied. Particles with the symmetry relaxed to either C10 or C11 were merged and subjected to a final round of auto-refinement. The distribution of all particles (%) and the resolution of each class is indicated. ( D ) FSC plot of the final refined undecameric unmasked (orange), masked (pink), phase-randomized (green) and corrected for mask convolution effects (blue) cryo-EM density map of CALHM4. The resolution at which the FSC curve drops below the 0.143 threshold is indicated. The inset shows the atomic model within the mask that was applied for calculations of the resolution estimates. ( E ) Final 3D reconstruction of undecameric CALHM4 colored according to local resolution. ( F ) FSC plot of the final refined decameric unmasked (orange), masked (pink), phase-randomized (green) and corrected for mask convolution effects (blue) cryo-EM density map of CALHM4. The resolution at which the FSC curve drops below the 0.143 threshold is indicated. The inset shows the atomic model within the mask that was applied for calculations of the resolution estimates. ( G ) Final 3D reconstruction of decameric CALHM4 colored according to local resolution.

Article Snippet: For structure determination of human CALHM2, 4 and 6 in the presence of Ca 2+ and of human CALHM4 in the absence of Ca 2+ by cryo-EM, 2.5 μl samples of GDN-purified proteins at a concentration of 1.5-3 mg ml −1 were applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3 or R0.6/1 Au 200 mesh).

Techniques: Cryo-EM Sample Prep, Microscopy

Journal: bioRxiv

Article Title: Cryo-EM structures and functional properties of CALHM channels of the human placenta

doi: 10.1101/2020.02.11.944231

Figure Lengend Snippet: Cryo-EM reconstruction of CALHM2 in presence of Ca 2+ . ( A ) Representative cryo-EM micrograph acquired with a Tecnai G 2 Polara microscope. ( B ) Data processing workflow. Non-symmetrized 3D classification with two reference models representing monomeric and dihedrally-related dimeric architectures as observed for CALHM6 and CALHM4, respectively, allowed to separate CALHM2 particles into two respective subsets. However, preferential orientation of the particles on a grid together with the presence of compositional heterogeneity in form of undecameric and dodecameric assemblies within each subset hindered generation of a high-resolution 3D reconstruction. 2D class averages calculated separately from monomeric and dimeric subsets highlight the predominance of views from the extracellular side.

Article Snippet: For structure determination of human CALHM2, 4 and 6 in the presence of Ca 2+ and of human CALHM4 in the absence of Ca 2+ by cryo-EM, 2.5 μl samples of GDN-purified proteins at a concentration of 1.5-3 mg ml −1 were applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3 or R0.6/1 Au 200 mesh).

Techniques: Cryo-EM Sample Prep, Microscopy

Journal: bioRxiv

Article Title: Cryo-EM structures and functional properties of CALHM channels of the human placenta

doi: 10.1101/2020.02.11.944231

Figure Lengend Snippet: Cryo-EM analysis. ( A ) Cryo-EM density of undecameric (11-mer) and decameric (10-mer) pairs of CALHM4 channels at 3.9 and 4.1 Å respectively. Data was recorded from a sample containing Ca 2+ . Subunits are colored in lilac and green, respectively. (B) Cryo-EM density of decameric CALHM6 channels at 4.5 Å. Subunits are colored in red and light-blue, respectively. A, B, Views are from within the membrane with membrane indicated as grey rectangle (top) and from the outside (bottom). ( C ) Selected 2D classes of the CALHM2 data showing interacting channel pairs and single channels viewed from within the membrane (top) and views of undecameric and dodecameric channels with subunits numbered (bottom). (D) Slices through the CALHM4 (left) and the CALHM6 (right) channels illustrating the distinct features of the cylindrical and conical pore conformations. View of CALHM6 at lower contour (right) shows extended density for the mobile TM1. Maps are low-pass filtered at 6 Å. Density corresponding to TM1 is colored according to A and B.

Article Snippet: For structure determination of human CALHM2, 4 and 6 in the presence of Ca 2+ and of human CALHM4 in the absence of Ca 2+ by cryo-EM, 2.5 μl samples of GDN-purified proteins at a concentration of 1.5-3 mg ml −1 were applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3 or R0.6/1 Au 200 mesh).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Cryo-EM structures and functional properties of CALHM channels of the human placenta

doi: 10.1101/2020.02.11.944231

Figure Lengend Snippet: Cryo-EM reconstruction of CALHM6 in presence of Ca 2+ . ( A ) Representative cryo-EM micrograph acquired with a Tecnai G 2 Polara microscope. ( B ) 2D class averages of CALHM6 in presence of Ca 2+ . ( C ) Data processing workflow. Non-symmetrized 3D classification allowed to isolate two populations representing decameric and undecameric assemblies. The particles were further refined with either C10 or C11 symmetry imposed and iterative per-particle CTF refinement and Bayesian polishing. The distribution of all particles (%) and the resolution of each class is indicated. ( D ) FSC plot of the final refined decameric unmasked (orange), masked (pink), phase-randomized (green) and corrected for mask convolution effects (blue) cryo-EM density map of CALHM6. The resolution at which the FSC curve drops below the 0.143 threshold is indicated. The inset shows the atomic model within the mask that was applied for calculations of the resolution estimates. ( E ) Final 3D reconstruction of decameric CALHM6 colored according to local resolution. ( F ) FSC plot of the final refined undecameric unmasked (orange), masked (pink), phase-randomized (green) and corrected for mask convolution effects (blue) cryo-EM density map of CALHM6. The resolution at which the FSC curve drops below the 0.143 threshold is indicated. The inset shows the atomic model within the mask that was applied for calculations of the resolution estimates. ( G ) Final 3D reconstruction of undecameric CALHM6 colored according to local resolution.

Article Snippet: For structure determination of human CALHM2, 4 and 6 in the presence of Ca 2+ and of human CALHM4 in the absence of Ca 2+ by cryo-EM, 2.5 μl samples of GDN-purified proteins at a concentration of 1.5-3 mg ml −1 were applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3 or R0.6/1 Au 200 mesh).

Techniques: Cryo-EM Sample Prep, Microscopy

Journal: bioRxiv

Article Title: Cryo-EM structures and functional properties of CALHM channels of the human placenta

doi: 10.1101/2020.02.11.944231

Figure Lengend Snippet: Cryo-EM reconstruction of CALHM4 in absence of Ca 2+ . ( A ) Representative cryo-EM micrograph acquired with Tecnai G 2 Polara microscope. ( B ) 2D class averages of CALHM4 in absence of Ca 2+ . ( C ) Data processing workflow. Two rounds of non-symmetrized 3D classification allowed to isolate two populations representing decameric and undecameric assemblies. The particles were further refined with either D10 or D11 symmetry imposed and iterative per-particle CTF refinement and Bayesian polishing. The distribution of all particles (%) and the resolution of each class is indicated. ( D ) FSC plot of the final refined undecameric unmasked (orange), masked (pink), phase-randomized (green) and corrected for mask convolution effects (blue) cryo-EM density map of CALHM4. The resolution at which the FSC curve drops below the 0.143 threshold is indicated. The inset shows the atomic model within the mask that was applied for calculations of the resolution estimates. ( E ) Final 3D reconstruction of undecameric CALHM4 colored according to local resolution. ( F ) FSC plot of the final refined decameric unmasked (orange), masked (pink), phase-randomized (green) and corrected for mask convolution effects (blue) cryo-EM density map of CALHM4. The resolution at which the FSC curve drops below the 0.143 threshold is indicated. The inset shows the atomic model within the mask that was applied for calculations of the resolution estimates. ( G ) Final 3D reconstruction of decameric CALHM4 colored according to local resolution.

Article Snippet: For structure determination of human CALHM2, 4 and 6 in the presence of Ca 2+ and of human CALHM4 in the absence of Ca 2+ by cryo-EM, 2.5 μl samples of GDN-purified proteins at a concentration of 1.5-3 mg ml −1 were applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3 or R0.6/1 Au 200 mesh).

Techniques: Cryo-EM Sample Prep, Microscopy

Journal: bioRxiv

Article Title: Cryo-EM structures and functional properties of CALHM channels of the human placenta

doi: 10.1101/2020.02.11.944231

Figure Lengend Snippet: Cryo-EM density of CALHM4 and CALHM6. ( A ) Cryo-EM density at 3.9 Å of selected regions of the undecameric CALHM4 structure recorded from a sample obtained in the presence of Ca 2+ superimposed on the model. Structural elements are indicated, ‘6 Å’ marks cryo-EM density low-pass filtered to 6 Å superimposed on a Cα trace of NH and TM1, which illustrates the helicity of the entire NH region that is partly not defined in the density at higher resolution due to its intrinsic mobility. ( B ) Cryo-EM density at 4.5 Å of selected regions of the decameric CALHM6 structure superimposed on the model. Structural elements are indicated. ‘6 Å’ marks cryo-EM density low-pass filtered to 6 Å superimposed on a ribbon of TM1 and parts of TM2, which displays extended density for the entire TM1 region that is partly not defined in the density at higher resolution due to its intrinsic mobility. Density is shown from two different views with indicated relationship. Large side chains that are recognizable in the low-pass filtered map are displayed as sticks.

Article Snippet: For structure determination of human CALHM2, 4 and 6 in the presence of Ca 2+ and of human CALHM4 in the absence of Ca 2+ by cryo-EM, 2.5 μl samples of GDN-purified proteins at a concentration of 1.5-3 mg ml −1 were applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3 or R0.6/1 Au 200 mesh).

Techniques: Cryo-EM Sample Prep