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Promega luminescent adp glo kinase assay system
Luminescent Adp Glo Kinase Assay System, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Promega adp glo assay kit
Adp Glo Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Promega adp glo kinase assay kit
Adp Glo Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Promega adp glo luminescence kinase assay
Adp Glo Luminescence Kinase Assay, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Promega adp glo kinase assay kit
Adp Glo Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/adp glo kinase assay kit/product/Promega
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Promega adp glo kinase assay kit
Adp Glo Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/adp glo kinase assay kit/product/Promega
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Promega promega adp glo assay
A: Histogram of rod diameters and the normalized abundance observed upon addition of <t>ADP</t> (red) during refolding in comparison <t>with</t> <t>PspA</t> apo (grey). In the presence of ADP, the distribution maximum shifts to 250 Å and diameters up to 400 Å could be observed. The associated cryo-EM structures are shown in the cross-sectional top and side views. B: Histogram of rod diameters upon addition of ATP (blue) during refolding in comparison with PspA apo (grey). In the presence of ATP, the rod population shifts further towards larger diameters in both cases. C: Corresponding three-dimensional cryo-EM atomic models. Frame highlights the most frequent structure for apo (215 Å, grey), ADP (250Å, red), and ATP (270 Å, blue) (α1 red, α2+3 violet, α4 blue, α5 cyan).
Promega Adp Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/promega adp glo assay/product/Promega
Average 86 stars, based on 1 article reviews
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promega adp glo assay - by Bioz Stars, 2024-07
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1) Product Images from "Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies"

Article Title: Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies

Journal: bioRxiv

doi: 10.1101/2024.07.08.602472

A: Histogram of rod diameters and the normalized abundance observed upon addition of ADP (red) during refolding in comparison with PspA apo (grey). In the presence of ADP, the distribution maximum shifts to 250 Å and diameters up to 400 Å could be observed. The associated cryo-EM structures are shown in the cross-sectional top and side views. B: Histogram of rod diameters upon addition of ATP (blue) during refolding in comparison with PspA apo (grey). In the presence of ATP, the rod population shifts further towards larger diameters in both cases. C: Corresponding three-dimensional cryo-EM atomic models. Frame highlights the most frequent structure for apo (215 Å, grey), ADP (250Å, red), and ATP (270 Å, blue) (α1 red, α2+3 violet, α4 blue, α5 cyan).
Figure Legend Snippet: A: Histogram of rod diameters and the normalized abundance observed upon addition of ADP (red) during refolding in comparison with PspA apo (grey). In the presence of ADP, the distribution maximum shifts to 250 Å and diameters up to 400 Å could be observed. The associated cryo-EM structures are shown in the cross-sectional top and side views. B: Histogram of rod diameters upon addition of ATP (blue) during refolding in comparison with PspA apo (grey). In the presence of ATP, the rod population shifts further towards larger diameters in both cases. C: Corresponding three-dimensional cryo-EM atomic models. Frame highlights the most frequent structure for apo (215 Å, grey), ADP (250Å, red), and ATP (270 Å, blue) (α1 red, α2+3 violet, α4 blue, α5 cyan).

Techniques Used: Comparison, Cryo-EM Sample Prep

Example micrographs of PspA samples. A: Cryo-EM micrographs of the different PspA data sets. Apo : PspA without additives. ATP: PspA with 2 mM ATP. ADP: PspA with 2 mM ADP; red arrows show “super rods” with engulfed “normal” rods. B: Sliced side and top view of the PspA cryo-EM density in the presence of ATP (200 Å diameter, PDB: 8AKR, EMDB: 15490) with a superimposed atomic PspA model. Putative nucleotide-binding sites are labeled with asterisks. Green arrows indicate solvent accessibility of the ATP binding site. Inset: Zoomed view of the putative nucleotide-binding site in the PspA rod structure (200 Å diameter) with weak segmented density above the loop connecting helices α3 and α4 (cyan) due to incomplete binding and imposition of helical symmetry. The nucleotide-binding site is formed by the surrounding chains (subunits j0, j-1, j+26, and j+27) and is repeated for every subunit in the polymer. Inset. View of the cryo-EM density of the nucleotide binding region superimposed with PspA, no nucleotide model included in density (black rim). C : Comparison of the PspA apo structure with PspA after incubation with ATP. Left column: Maps of PspA and PspA+ATP at low and high contour levels superimposed. Middle column: Models of PspA apo (PDB:7ABK) and PspA+ATP with an ADP model fitted to the additional density. Note that due to the presence of the nucleotide the loop in the PspA+ATP structure is moved. Right column: Atomic models of PspA apo and PspA+ATP superimposed with colored atomic temperature factors indicate increased mobility of α3/α4 loop in the PspA apo structure.
Figure Legend Snippet: Example micrographs of PspA samples. A: Cryo-EM micrographs of the different PspA data sets. Apo : PspA without additives. ATP: PspA with 2 mM ATP. ADP: PspA with 2 mM ADP; red arrows show “super rods” with engulfed “normal” rods. B: Sliced side and top view of the PspA cryo-EM density in the presence of ATP (200 Å diameter, PDB: 8AKR, EMDB: 15490) with a superimposed atomic PspA model. Putative nucleotide-binding sites are labeled with asterisks. Green arrows indicate solvent accessibility of the ATP binding site. Inset: Zoomed view of the putative nucleotide-binding site in the PspA rod structure (200 Å diameter) with weak segmented density above the loop connecting helices α3 and α4 (cyan) due to incomplete binding and imposition of helical symmetry. The nucleotide-binding site is formed by the surrounding chains (subunits j0, j-1, j+26, and j+27) and is repeated for every subunit in the polymer. Inset. View of the cryo-EM density of the nucleotide binding region superimposed with PspA, no nucleotide model included in density (black rim). C : Comparison of the PspA apo structure with PspA after incubation with ATP. Left column: Maps of PspA and PspA+ATP at low and high contour levels superimposed. Middle column: Models of PspA apo (PDB:7ABK) and PspA+ATP with an ADP model fitted to the additional density. Note that due to the presence of the nucleotide the loop in the PspA+ATP structure is moved. Right column: Atomic models of PspA apo and PspA+ATP superimposed with colored atomic temperature factors indicate increased mobility of α3/α4 loop in the PspA apo structure.

Techniques Used: Cryo-EM Sample Prep, Binding Assay, Labeling, Solvent, Polymer, Comparison, Incubation

PspA rod diameter distribution for A: PspA apo (grey), C: PspA+ADP (red), and E: PspA+ATP (blue), based on the relative occurrence of rod segments with a certain diameter. B, D , F: Overview of PspA rod cryo-EM structures with cross-sectional top view z -slices (left column), cross-sectional side view xy -slices (middle column) and FSC curves with a 0.143 threshold of B: PspA apo , D: PspA+ADP and F: PspA+ATP. Note that when FSC curves drop below 1, systematic peaks of high correlation occur at 1/5.3 Å corresponding to the α-helical pitch feature and additional low-resolution details of the all α-helical maps of PspA. G: Comparison of the PspA ATP 200 Å monomer with the high-resolution reference structure of PspA apo PDB:7ABK.
Figure Legend Snippet: PspA rod diameter distribution for A: PspA apo (grey), C: PspA+ADP (red), and E: PspA+ATP (blue), based on the relative occurrence of rod segments with a certain diameter. B, D , F: Overview of PspA rod cryo-EM structures with cross-sectional top view z -slices (left column), cross-sectional side view xy -slices (middle column) and FSC curves with a 0.143 threshold of B: PspA apo , D: PspA+ADP and F: PspA+ATP. Note that when FSC curves drop below 1, systematic peaks of high correlation occur at 1/5.3 Å corresponding to the α-helical pitch feature and additional low-resolution details of the all α-helical maps of PspA. G: Comparison of the PspA ATP 200 Å monomer with the high-resolution reference structure of PspA apo PDB:7ABK.

Techniques Used: Cryo-EM Sample Prep, Comparison

PspA rod radial density profiles and mass per length. A: Cryo-EM density fit of atomic PspA models of different diameters focusing on the helices α1/α2 hairpin and helix 5 ((1 red, (2+3 violet, (4 blue, (5 cyan). (180 Å: PDB 8AKQ, 200 Å: PDB 8AKR, 215 Å: PDB 8AKS, 235 Å: PDB 8AKT, 250 Å: PDB 8AKU, 270 Å: PDB 8AKV, 280 Å: PDB 8AKW, 290 Å: PDB 8AKY, 305 Å: PDB 8AKX, 320 Å: PDB 8AKZ, 365 Å: PDB 8AL0) B: Scatter plot of three selected pairs (R44-L185, E126-S163, E179-K55) distance changes with respect to the initial distance in the 180 Å diameter assembly over all rod diameters. C: Box plot of pair distance changes of evolutionarily conserved residues with respect to the initial distance in the 180 Å diameter assembly. The residues were selected by first identifying potential intermolecular interactions between highly conserved residues (>90% conserved among PspA/Vipp1proteins . Then, the C( distance for each pair was measured for each rod diameter. To calculate the difference of the pair distances relative to the smallest diameter rods, the distances in 180 Å rods were subtracted from the respective distances in the other diameters. The standard deviation (SD) of the distance shift distribution is a measure of the flexibility of the interaction. Boxes show SD with median line (line) and mean value (circle). Whiskers show the range within 1.5 IQR. Color code for residues: green=charged; yellow=hydrophobic; pink=polar+uncharged: grey=special cases. Color code for boxes: residues located in helix α1: red, residues located in helix α2+3: violet, residues located in helix α4: blue, residues located in helix α5: cyan. D: Diameters plotted against helical rise (after correction for number of strands) in Å. Apo (black): PspA ADP (red): PspA + 2mM ADP. ATP (blue): PspA + 2 mM ATP. E: Diameters plotted against mass per 100 Å length in kDa. Apo (black): PspA . ADP (red): PspA + 2mM ADP. ATP (blue): PspA + 2 mM ATP. F: PspA models with 60 monomers each show a decrease in rod length and an increasing diameter from 180 - 365 Å.
Figure Legend Snippet: PspA rod radial density profiles and mass per length. A: Cryo-EM density fit of atomic PspA models of different diameters focusing on the helices α1/α2 hairpin and helix 5 ((1 red, (2+3 violet, (4 blue, (5 cyan). (180 Å: PDB 8AKQ, 200 Å: PDB 8AKR, 215 Å: PDB 8AKS, 235 Å: PDB 8AKT, 250 Å: PDB 8AKU, 270 Å: PDB 8AKV, 280 Å: PDB 8AKW, 290 Å: PDB 8AKY, 305 Å: PDB 8AKX, 320 Å: PDB 8AKZ, 365 Å: PDB 8AL0) B: Scatter plot of three selected pairs (R44-L185, E126-S163, E179-K55) distance changes with respect to the initial distance in the 180 Å diameter assembly over all rod diameters. C: Box plot of pair distance changes of evolutionarily conserved residues with respect to the initial distance in the 180 Å diameter assembly. The residues were selected by first identifying potential intermolecular interactions between highly conserved residues (>90% conserved among PspA/Vipp1proteins . Then, the C( distance for each pair was measured for each rod diameter. To calculate the difference of the pair distances relative to the smallest diameter rods, the distances in 180 Å rods were subtracted from the respective distances in the other diameters. The standard deviation (SD) of the distance shift distribution is a measure of the flexibility of the interaction. Boxes show SD with median line (line) and mean value (circle). Whiskers show the range within 1.5 IQR. Color code for residues: green=charged; yellow=hydrophobic; pink=polar+uncharged: grey=special cases. Color code for boxes: residues located in helix α1: red, residues located in helix α2+3: violet, residues located in helix α4: blue, residues located in helix α5: cyan. D: Diameters plotted against helical rise (after correction for number of strands) in Å. Apo (black): PspA ADP (red): PspA + 2mM ADP. ATP (blue): PspA + 2 mM ATP. E: Diameters plotted against mass per 100 Å length in kDa. Apo (black): PspA . ADP (red): PspA + 2mM ADP. ATP (blue): PspA + 2 mM ATP. F: PspA models with 60 monomers each show a decrease in rod length and an increasing diameter from 180 - 365 Å.

Techniques Used: Cryo-EM Sample Prep, Standard Deviation

A: Schematic secondary structure topology of the PspA monomer (α1 red, α2+3 violet, α4 blue, α5 cyan). Relevant mutations are indicated in green. B: ATPase activity of PspA wild type (WT) and mutants measured by a malachite-green based assay. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. p- values of two-sample t-test: p(WT_vs R44K/E126Q/E179Q) =7.18*10 -9 , p(WT_vs α1-5) =0.63 (n(WT)=36, n(R44K)=24, n(E126Q)=27, n(E179Q)=18, n(R44K/E126Q)=21, n(R44K/E179Q)=18, n(E126Q/E179Q)=18, n(R44K/E126Q/E179Q)=21, n(α1-5)=18; For all measurements, samples of at least 3 biological replicates were included. C: ATPase activity of the WT protein and the α1-5 mutant without and in the presence of EPL; WT (orange): ATPase activity without EPL; WT EPL (green): ATPase activity in the presence of EPL (SUVs made from E. coli polar lipid extract); α1-5 variant (purple); n=3. D: HPLC/MS-MS of PspA + ATP after 24 h incubation and extensive washing. Different color lines represent different MRM transitions. For ADP, MRM transitions are 426/134 (red) and 426/159 (light red). For ATP MRM transitions are 506/79 (dark grey) and 506/159 (light grey). The ADP fragments below the ATP peak are formed by in-source fragmentation of ATP in the ESI source. E: Negative staining EM micrographs of PspA WT protein and PspA mutants. Magnification 57 kx.
Figure Legend Snippet: A: Schematic secondary structure topology of the PspA monomer (α1 red, α2+3 violet, α4 blue, α5 cyan). Relevant mutations are indicated in green. B: ATPase activity of PspA wild type (WT) and mutants measured by a malachite-green based assay. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. p- values of two-sample t-test: p(WT_vs R44K/E126Q/E179Q) =7.18*10 -9 , p(WT_vs α1-5) =0.63 (n(WT)=36, n(R44K)=24, n(E126Q)=27, n(E179Q)=18, n(R44K/E126Q)=21, n(R44K/E179Q)=18, n(E126Q/E179Q)=18, n(R44K/E126Q/E179Q)=21, n(α1-5)=18; For all measurements, samples of at least 3 biological replicates were included. C: ATPase activity of the WT protein and the α1-5 mutant without and in the presence of EPL; WT (orange): ATPase activity without EPL; WT EPL (green): ATPase activity in the presence of EPL (SUVs made from E. coli polar lipid extract); α1-5 variant (purple); n=3. D: HPLC/MS-MS of PspA + ATP after 24 h incubation and extensive washing. Different color lines represent different MRM transitions. For ADP, MRM transitions are 426/134 (red) and 426/159 (light red). For ATP MRM transitions are 506/79 (dark grey) and 506/159 (light grey). The ADP fragments below the ATP peak are formed by in-source fragmentation of ATP in the ESI source. E: Negative staining EM micrographs of PspA WT protein and PspA mutants. Magnification 57 kx.

Techniques Used: Activity Assay, Malachite Green Assay, Mutagenesis, Variant Assay, Tandem Mass Spectroscopy, Incubation, Negative Staining

A: SDS PAGE of purified PspA WT and mutants (3 µg each). Purified proteins show a band at 28 kDa, except for the α0 truncated form which shows a band at 25 kDa. Asterisk: Faint dimer bands at twice the height of the monomeric protein band (56 kDa for WT and full-length PspA mutants; 50 kDa for α1-5 mutant). Marker: Bio-Rad Precision PlusProteinTM Unstained standards. B: ATPase activity of PspA. Left graph: Influence of Mg 2+ and EDTA on the ATPase activity of the WT protein. WT (orange): ATPase activity in the presence of 2 mM Mg 2+ . -Mg 2+ (green): ATPase activity in the absence of Mg 2+ . EDTA (purple): ATPase activity in the presence of 10 mM EDTA; n(WT)=36, n(-Mg 2+ and EDTA)=3. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. Middle graph: Comparison of the PspA ATPase activity with ATPase activity of E. coli PspF, n(PspA)=36, n( Eco PspF)=3. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. Right graph: Influence of ATPyS and AMPPCP on the ATPase activity of PspA. Red circle: ATPyS; Grey square: AMPPCP; n=3, error bars represent SD. C: HPLC/MS-MS of PspA directly after purification. D: HPLC/MS-MS of PspA R44K/E126Q/E179Q + ATP after 24 h incubation and extensive washing. Different color lines represent different MRM transitions. For ADP, MRM transitions are 426/134 (red) and 426/159 (light red). For ATP MRM transitions are 506/79 (dark grey) and 506/159 (light grey). The ADP fragments below the ATP peak are formed by in-source fragmentation of ATP in the ESI source. E: Bar plot of estimated nucleotide concentrations found by LC-MS/MS in PspA apo control, WT, and R44K/E126Q/E179Q rods after incubation with ATP for 24 h and extensive washing, n=1.
Figure Legend Snippet: A: SDS PAGE of purified PspA WT and mutants (3 µg each). Purified proteins show a band at 28 kDa, except for the α0 truncated form which shows a band at 25 kDa. Asterisk: Faint dimer bands at twice the height of the monomeric protein band (56 kDa for WT and full-length PspA mutants; 50 kDa for α1-5 mutant). Marker: Bio-Rad Precision PlusProteinTM Unstained standards. B: ATPase activity of PspA. Left graph: Influence of Mg 2+ and EDTA on the ATPase activity of the WT protein. WT (orange): ATPase activity in the presence of 2 mM Mg 2+ . -Mg 2+ (green): ATPase activity in the absence of Mg 2+ . EDTA (purple): ATPase activity in the presence of 10 mM EDTA; n(WT)=36, n(-Mg 2+ and EDTA)=3. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. Middle graph: Comparison of the PspA ATPase activity with ATPase activity of E. coli PspF, n(PspA)=36, n( Eco PspF)=3. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. Right graph: Influence of ATPyS and AMPPCP on the ATPase activity of PspA. Red circle: ATPyS; Grey square: AMPPCP; n=3, error bars represent SD. C: HPLC/MS-MS of PspA directly after purification. D: HPLC/MS-MS of PspA R44K/E126Q/E179Q + ATP after 24 h incubation and extensive washing. Different color lines represent different MRM transitions. For ADP, MRM transitions are 426/134 (red) and 426/159 (light red). For ATP MRM transitions are 506/79 (dark grey) and 506/159 (light grey). The ADP fragments below the ATP peak are formed by in-source fragmentation of ATP in the ESI source. E: Bar plot of estimated nucleotide concentrations found by LC-MS/MS in PspA apo control, WT, and R44K/E126Q/E179Q rods after incubation with ATP for 24 h and extensive washing, n=1.

Techniques Used: SDS Page, Purification, Mutagenesis, Marker, Activity Assay, Comparison, Tandem Mass Spectroscopy, Incubation, Liquid Chromatography with Mass Spectroscopy, Control

Top. PspA rod diameter distribution for A: PspA+ATP (blue), and preformed PspA rods incubated with ATP (brown), C: PspA+ADP (red) and PspA+AMPPCP (magenta), and E: PspA+ATP (blue) and PspA+ATPyS (cyan), based on the relative occurrence of rod segments with a certain diameter. Bottom. Cross-sectional top view z -slices (left column), cross-sectional side view xy -slices (middle column) and FSC curves (threshold 0.143) of cryo-EM structures B: PspA+ATP (preformed), D: PspA+AMPPCP and F: PspA+ATPyS. G: FSC curve of PspA dL10 diameters (threshold 0.143). H: FSC curve of PspA dL10 diameters with ATP (threshold 0.143). Note that when FSC curves drop below 1, systematic peaks of high correlation occur at 1/5.3 Å corresponding to the α-helical pitch and additional low-resolution features of the all α-helical maps of PspA.
Figure Legend Snippet: Top. PspA rod diameter distribution for A: PspA+ATP (blue), and preformed PspA rods incubated with ATP (brown), C: PspA+ADP (red) and PspA+AMPPCP (magenta), and E: PspA+ATP (blue) and PspA+ATPyS (cyan), based on the relative occurrence of rod segments with a certain diameter. Bottom. Cross-sectional top view z -slices (left column), cross-sectional side view xy -slices (middle column) and FSC curves (threshold 0.143) of cryo-EM structures B: PspA+ATP (preformed), D: PspA+AMPPCP and F: PspA+ATPyS. G: FSC curve of PspA dL10 diameters (threshold 0.143). H: FSC curve of PspA dL10 diameters with ATP (threshold 0.143). Note that when FSC curves drop below 1, systematic peaks of high correlation occur at 1/5.3 Å corresponding to the α-helical pitch and additional low-resolution features of the all α-helical maps of PspA.

Techniques Used: Incubation, Cryo-EM Sample Prep


Structured Review

Promega adp glo kinase assay kit
Adp Glo Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/adp glo kinase assay kit/product/Promega
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
adp glo kinase assay kit - by Bioz Stars, 2024-07
86/100 stars

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Structured Review

Promega adp glo kinase assays
Adp Glo Kinase Assays, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/adp glo kinase assays/product/Promega
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
adp glo kinase assays - by Bioz Stars, 2024-07
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Structured Review

Promega adp glo kinase assays
Adp Glo Kinase Assays, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/adp glo kinase assays/product/Promega
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
adp glo kinase assays - by Bioz Stars, 2024-07
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    A: Histogram of rod diameters and the normalized abundance observed upon addition of <t>ADP</t> (red) during refolding in comparison <t>with</t> <t>PspA</t> apo (grey). In the presence of ADP, the distribution maximum shifts to 250 Å and diameters up to 400 Å could be observed. The associated cryo-EM structures are shown in the cross-sectional top and side views. B: Histogram of rod diameters upon addition of ATP (blue) during refolding in comparison with PspA apo (grey). In the presence of ATP, the rod population shifts further towards larger diameters in both cases. C: Corresponding three-dimensional cryo-EM atomic models. Frame highlights the most frequent structure for apo (215 Å, grey), ADP (250Å, red), and ATP (270 Å, blue) (α1 red, α2+3 violet, α4 blue, α5 cyan).
    Promega Adp Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega adp glo kinase assays
    A: Histogram of rod diameters and the normalized abundance observed upon addition of <t>ADP</t> (red) during refolding in comparison <t>with</t> <t>PspA</t> apo (grey). In the presence of ADP, the distribution maximum shifts to 250 Å and diameters up to 400 Å could be observed. The associated cryo-EM structures are shown in the cross-sectional top and side views. B: Histogram of rod diameters upon addition of ATP (blue) during refolding in comparison with PspA apo (grey). In the presence of ATP, the rod population shifts further towards larger diameters in both cases. C: Corresponding three-dimensional cryo-EM atomic models. Frame highlights the most frequent structure for apo (215 Å, grey), ADP (250Å, red), and ATP (270 Å, blue) (α1 red, α2+3 violet, α4 blue, α5 cyan).
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    A: Histogram of rod diameters and the normalized abundance observed upon addition of ADP (red) during refolding in comparison with PspA apo (grey). In the presence of ADP, the distribution maximum shifts to 250 Å and diameters up to 400 Å could be observed. The associated cryo-EM structures are shown in the cross-sectional top and side views. B: Histogram of rod diameters upon addition of ATP (blue) during refolding in comparison with PspA apo (grey). In the presence of ATP, the rod population shifts further towards larger diameters in both cases. C: Corresponding three-dimensional cryo-EM atomic models. Frame highlights the most frequent structure for apo (215 Å, grey), ADP (250Å, red), and ATP (270 Å, blue) (α1 red, α2+3 violet, α4 blue, α5 cyan).

    Journal: bioRxiv

    Article Title: Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies

    doi: 10.1101/2024.07.08.602472

    Figure Lengend Snippet: A: Histogram of rod diameters and the normalized abundance observed upon addition of ADP (red) during refolding in comparison with PspA apo (grey). In the presence of ADP, the distribution maximum shifts to 250 Å and diameters up to 400 Å could be observed. The associated cryo-EM structures are shown in the cross-sectional top and side views. B: Histogram of rod diameters upon addition of ATP (blue) during refolding in comparison with PspA apo (grey). In the presence of ATP, the rod population shifts further towards larger diameters in both cases. C: Corresponding three-dimensional cryo-EM atomic models. Frame highlights the most frequent structure for apo (215 Å, grey), ADP (250Å, red), and ATP (270 Å, blue) (α1 red, α2+3 violet, α4 blue, α5 cyan).

    Article Snippet: The ATPase activity of PspA was calculated as: with the average ATPase activity A ATPase , the phosphate release in µM ϕ[P i ] , the protein concentration in µM [Protein], and the incubation time of the reaction in hours t . To determine the ATPase activity of PspA in the presence of lipids, the ADP formed during the ATPase reaction of PspA was measured using a Promega ADP Glo Assay.

    Techniques: Comparison, Cryo-EM Sample Prep

    Example micrographs of PspA samples. A: Cryo-EM micrographs of the different PspA data sets. Apo : PspA without additives. ATP: PspA with 2 mM ATP. ADP: PspA with 2 mM ADP; red arrows show “super rods” with engulfed “normal” rods. B: Sliced side and top view of the PspA cryo-EM density in the presence of ATP (200 Å diameter, PDB: 8AKR, EMDB: 15490) with a superimposed atomic PspA model. Putative nucleotide-binding sites are labeled with asterisks. Green arrows indicate solvent accessibility of the ATP binding site. Inset: Zoomed view of the putative nucleotide-binding site in the PspA rod structure (200 Å diameter) with weak segmented density above the loop connecting helices α3 and α4 (cyan) due to incomplete binding and imposition of helical symmetry. The nucleotide-binding site is formed by the surrounding chains (subunits j0, j-1, j+26, and j+27) and is repeated for every subunit in the polymer. Inset. View of the cryo-EM density of the nucleotide binding region superimposed with PspA, no nucleotide model included in density (black rim). C : Comparison of the PspA apo structure with PspA after incubation with ATP. Left column: Maps of PspA and PspA+ATP at low and high contour levels superimposed. Middle column: Models of PspA apo (PDB:7ABK) and PspA+ATP with an ADP model fitted to the additional density. Note that due to the presence of the nucleotide the loop in the PspA+ATP structure is moved. Right column: Atomic models of PspA apo and PspA+ATP superimposed with colored atomic temperature factors indicate increased mobility of α3/α4 loop in the PspA apo structure.

    Journal: bioRxiv

    Article Title: Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies

    doi: 10.1101/2024.07.08.602472

    Figure Lengend Snippet: Example micrographs of PspA samples. A: Cryo-EM micrographs of the different PspA data sets. Apo : PspA without additives. ATP: PspA with 2 mM ATP. ADP: PspA with 2 mM ADP; red arrows show “super rods” with engulfed “normal” rods. B: Sliced side and top view of the PspA cryo-EM density in the presence of ATP (200 Å diameter, PDB: 8AKR, EMDB: 15490) with a superimposed atomic PspA model. Putative nucleotide-binding sites are labeled with asterisks. Green arrows indicate solvent accessibility of the ATP binding site. Inset: Zoomed view of the putative nucleotide-binding site in the PspA rod structure (200 Å diameter) with weak segmented density above the loop connecting helices α3 and α4 (cyan) due to incomplete binding and imposition of helical symmetry. The nucleotide-binding site is formed by the surrounding chains (subunits j0, j-1, j+26, and j+27) and is repeated for every subunit in the polymer. Inset. View of the cryo-EM density of the nucleotide binding region superimposed with PspA, no nucleotide model included in density (black rim). C : Comparison of the PspA apo structure with PspA after incubation with ATP. Left column: Maps of PspA and PspA+ATP at low and high contour levels superimposed. Middle column: Models of PspA apo (PDB:7ABK) and PspA+ATP with an ADP model fitted to the additional density. Note that due to the presence of the nucleotide the loop in the PspA+ATP structure is moved. Right column: Atomic models of PspA apo and PspA+ATP superimposed with colored atomic temperature factors indicate increased mobility of α3/α4 loop in the PspA apo structure.

    Article Snippet: The ATPase activity of PspA was calculated as: with the average ATPase activity A ATPase , the phosphate release in µM ϕ[P i ] , the protein concentration in µM [Protein], and the incubation time of the reaction in hours t . To determine the ATPase activity of PspA in the presence of lipids, the ADP formed during the ATPase reaction of PspA was measured using a Promega ADP Glo Assay.

    Techniques: Cryo-EM Sample Prep, Binding Assay, Labeling, Solvent, Polymer, Comparison, Incubation

    PspA rod diameter distribution for A: PspA apo (grey), C: PspA+ADP (red), and E: PspA+ATP (blue), based on the relative occurrence of rod segments with a certain diameter. B, D , F: Overview of PspA rod cryo-EM structures with cross-sectional top view z -slices (left column), cross-sectional side view xy -slices (middle column) and FSC curves with a 0.143 threshold of B: PspA apo , D: PspA+ADP and F: PspA+ATP. Note that when FSC curves drop below 1, systematic peaks of high correlation occur at 1/5.3 Å corresponding to the α-helical pitch feature and additional low-resolution details of the all α-helical maps of PspA. G: Comparison of the PspA ATP 200 Å monomer with the high-resolution reference structure of PspA apo PDB:7ABK.

    Journal: bioRxiv

    Article Title: Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies

    doi: 10.1101/2024.07.08.602472

    Figure Lengend Snippet: PspA rod diameter distribution for A: PspA apo (grey), C: PspA+ADP (red), and E: PspA+ATP (blue), based on the relative occurrence of rod segments with a certain diameter. B, D , F: Overview of PspA rod cryo-EM structures with cross-sectional top view z -slices (left column), cross-sectional side view xy -slices (middle column) and FSC curves with a 0.143 threshold of B: PspA apo , D: PspA+ADP and F: PspA+ATP. Note that when FSC curves drop below 1, systematic peaks of high correlation occur at 1/5.3 Å corresponding to the α-helical pitch feature and additional low-resolution details of the all α-helical maps of PspA. G: Comparison of the PspA ATP 200 Å monomer with the high-resolution reference structure of PspA apo PDB:7ABK.

    Article Snippet: The ATPase activity of PspA was calculated as: with the average ATPase activity A ATPase , the phosphate release in µM ϕ[P i ] , the protein concentration in µM [Protein], and the incubation time of the reaction in hours t . To determine the ATPase activity of PspA in the presence of lipids, the ADP formed during the ATPase reaction of PspA was measured using a Promega ADP Glo Assay.

    Techniques: Cryo-EM Sample Prep, Comparison

    PspA rod radial density profiles and mass per length. A: Cryo-EM density fit of atomic PspA models of different diameters focusing on the helices α1/α2 hairpin and helix 5 ((1 red, (2+3 violet, (4 blue, (5 cyan). (180 Å: PDB 8AKQ, 200 Å: PDB 8AKR, 215 Å: PDB 8AKS, 235 Å: PDB 8AKT, 250 Å: PDB 8AKU, 270 Å: PDB 8AKV, 280 Å: PDB 8AKW, 290 Å: PDB 8AKY, 305 Å: PDB 8AKX, 320 Å: PDB 8AKZ, 365 Å: PDB 8AL0) B: Scatter plot of three selected pairs (R44-L185, E126-S163, E179-K55) distance changes with respect to the initial distance in the 180 Å diameter assembly over all rod diameters. C: Box plot of pair distance changes of evolutionarily conserved residues with respect to the initial distance in the 180 Å diameter assembly. The residues were selected by first identifying potential intermolecular interactions between highly conserved residues (>90% conserved among PspA/Vipp1proteins . Then, the C( distance for each pair was measured for each rod diameter. To calculate the difference of the pair distances relative to the smallest diameter rods, the distances in 180 Å rods were subtracted from the respective distances in the other diameters. The standard deviation (SD) of the distance shift distribution is a measure of the flexibility of the interaction. Boxes show SD with median line (line) and mean value (circle). Whiskers show the range within 1.5 IQR. Color code for residues: green=charged; yellow=hydrophobic; pink=polar+uncharged: grey=special cases. Color code for boxes: residues located in helix α1: red, residues located in helix α2+3: violet, residues located in helix α4: blue, residues located in helix α5: cyan. D: Diameters plotted against helical rise (after correction for number of strands) in Å. Apo (black): PspA ADP (red): PspA + 2mM ADP. ATP (blue): PspA + 2 mM ATP. E: Diameters plotted against mass per 100 Å length in kDa. Apo (black): PspA . ADP (red): PspA + 2mM ADP. ATP (blue): PspA + 2 mM ATP. F: PspA models with 60 monomers each show a decrease in rod length and an increasing diameter from 180 - 365 Å.

    Journal: bioRxiv

    Article Title: Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies

    doi: 10.1101/2024.07.08.602472

    Figure Lengend Snippet: PspA rod radial density profiles and mass per length. A: Cryo-EM density fit of atomic PspA models of different diameters focusing on the helices α1/α2 hairpin and helix 5 ((1 red, (2+3 violet, (4 blue, (5 cyan). (180 Å: PDB 8AKQ, 200 Å: PDB 8AKR, 215 Å: PDB 8AKS, 235 Å: PDB 8AKT, 250 Å: PDB 8AKU, 270 Å: PDB 8AKV, 280 Å: PDB 8AKW, 290 Å: PDB 8AKY, 305 Å: PDB 8AKX, 320 Å: PDB 8AKZ, 365 Å: PDB 8AL0) B: Scatter plot of three selected pairs (R44-L185, E126-S163, E179-K55) distance changes with respect to the initial distance in the 180 Å diameter assembly over all rod diameters. C: Box plot of pair distance changes of evolutionarily conserved residues with respect to the initial distance in the 180 Å diameter assembly. The residues were selected by first identifying potential intermolecular interactions between highly conserved residues (>90% conserved among PspA/Vipp1proteins . Then, the C( distance for each pair was measured for each rod diameter. To calculate the difference of the pair distances relative to the smallest diameter rods, the distances in 180 Å rods were subtracted from the respective distances in the other diameters. The standard deviation (SD) of the distance shift distribution is a measure of the flexibility of the interaction. Boxes show SD with median line (line) and mean value (circle). Whiskers show the range within 1.5 IQR. Color code for residues: green=charged; yellow=hydrophobic; pink=polar+uncharged: grey=special cases. Color code for boxes: residues located in helix α1: red, residues located in helix α2+3: violet, residues located in helix α4: blue, residues located in helix α5: cyan. D: Diameters plotted against helical rise (after correction for number of strands) in Å. Apo (black): PspA ADP (red): PspA + 2mM ADP. ATP (blue): PspA + 2 mM ATP. E: Diameters plotted against mass per 100 Å length in kDa. Apo (black): PspA . ADP (red): PspA + 2mM ADP. ATP (blue): PspA + 2 mM ATP. F: PspA models with 60 monomers each show a decrease in rod length and an increasing diameter from 180 - 365 Å.

    Article Snippet: The ATPase activity of PspA was calculated as: with the average ATPase activity A ATPase , the phosphate release in µM ϕ[P i ] , the protein concentration in µM [Protein], and the incubation time of the reaction in hours t . To determine the ATPase activity of PspA in the presence of lipids, the ADP formed during the ATPase reaction of PspA was measured using a Promega ADP Glo Assay.

    Techniques: Cryo-EM Sample Prep, Standard Deviation

    A: Schematic secondary structure topology of the PspA monomer (α1 red, α2+3 violet, α4 blue, α5 cyan). Relevant mutations are indicated in green. B: ATPase activity of PspA wild type (WT) and mutants measured by a malachite-green based assay. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. p- values of two-sample t-test: p(WT_vs R44K/E126Q/E179Q) =7.18*10 -9 , p(WT_vs α1-5) =0.63 (n(WT)=36, n(R44K)=24, n(E126Q)=27, n(E179Q)=18, n(R44K/E126Q)=21, n(R44K/E179Q)=18, n(E126Q/E179Q)=18, n(R44K/E126Q/E179Q)=21, n(α1-5)=18; For all measurements, samples of at least 3 biological replicates were included. C: ATPase activity of the WT protein and the α1-5 mutant without and in the presence of EPL; WT (orange): ATPase activity without EPL; WT EPL (green): ATPase activity in the presence of EPL (SUVs made from E. coli polar lipid extract); α1-5 variant (purple); n=3. D: HPLC/MS-MS of PspA + ATP after 24 h incubation and extensive washing. Different color lines represent different MRM transitions. For ADP, MRM transitions are 426/134 (red) and 426/159 (light red). For ATP MRM transitions are 506/79 (dark grey) and 506/159 (light grey). The ADP fragments below the ATP peak are formed by in-source fragmentation of ATP in the ESI source. E: Negative staining EM micrographs of PspA WT protein and PspA mutants. Magnification 57 kx.

    Journal: bioRxiv

    Article Title: Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies

    doi: 10.1101/2024.07.08.602472

    Figure Lengend Snippet: A: Schematic secondary structure topology of the PspA monomer (α1 red, α2+3 violet, α4 blue, α5 cyan). Relevant mutations are indicated in green. B: ATPase activity of PspA wild type (WT) and mutants measured by a malachite-green based assay. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. p- values of two-sample t-test: p(WT_vs R44K/E126Q/E179Q) =7.18*10 -9 , p(WT_vs α1-5) =0.63 (n(WT)=36, n(R44K)=24, n(E126Q)=27, n(E179Q)=18, n(R44K/E126Q)=21, n(R44K/E179Q)=18, n(E126Q/E179Q)=18, n(R44K/E126Q/E179Q)=21, n(α1-5)=18; For all measurements, samples of at least 3 biological replicates were included. C: ATPase activity of the WT protein and the α1-5 mutant without and in the presence of EPL; WT (orange): ATPase activity without EPL; WT EPL (green): ATPase activity in the presence of EPL (SUVs made from E. coli polar lipid extract); α1-5 variant (purple); n=3. D: HPLC/MS-MS of PspA + ATP after 24 h incubation and extensive washing. Different color lines represent different MRM transitions. For ADP, MRM transitions are 426/134 (red) and 426/159 (light red). For ATP MRM transitions are 506/79 (dark grey) and 506/159 (light grey). The ADP fragments below the ATP peak are formed by in-source fragmentation of ATP in the ESI source. E: Negative staining EM micrographs of PspA WT protein and PspA mutants. Magnification 57 kx.

    Article Snippet: The ATPase activity of PspA was calculated as: with the average ATPase activity A ATPase , the phosphate release in µM ϕ[P i ] , the protein concentration in µM [Protein], and the incubation time of the reaction in hours t . To determine the ATPase activity of PspA in the presence of lipids, the ADP formed during the ATPase reaction of PspA was measured using a Promega ADP Glo Assay.

    Techniques: Activity Assay, Malachite Green Assay, Mutagenesis, Variant Assay, Tandem Mass Spectroscopy, Incubation, Negative Staining

    A: SDS PAGE of purified PspA WT and mutants (3 µg each). Purified proteins show a band at 28 kDa, except for the α0 truncated form which shows a band at 25 kDa. Asterisk: Faint dimer bands at twice the height of the monomeric protein band (56 kDa for WT and full-length PspA mutants; 50 kDa for α1-5 mutant). Marker: Bio-Rad Precision PlusProteinTM Unstained standards. B: ATPase activity of PspA. Left graph: Influence of Mg 2+ and EDTA on the ATPase activity of the WT protein. WT (orange): ATPase activity in the presence of 2 mM Mg 2+ . -Mg 2+ (green): ATPase activity in the absence of Mg 2+ . EDTA (purple): ATPase activity in the presence of 10 mM EDTA; n(WT)=36, n(-Mg 2+ and EDTA)=3. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. Middle graph: Comparison of the PspA ATPase activity with ATPase activity of E. coli PspF, n(PspA)=36, n( Eco PspF)=3. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. Right graph: Influence of ATPyS and AMPPCP on the ATPase activity of PspA. Red circle: ATPyS; Grey square: AMPPCP; n=3, error bars represent SD. C: HPLC/MS-MS of PspA directly after purification. D: HPLC/MS-MS of PspA R44K/E126Q/E179Q + ATP after 24 h incubation and extensive washing. Different color lines represent different MRM transitions. For ADP, MRM transitions are 426/134 (red) and 426/159 (light red). For ATP MRM transitions are 506/79 (dark grey) and 506/159 (light grey). The ADP fragments below the ATP peak are formed by in-source fragmentation of ATP in the ESI source. E: Bar plot of estimated nucleotide concentrations found by LC-MS/MS in PspA apo control, WT, and R44K/E126Q/E179Q rods after incubation with ATP for 24 h and extensive washing, n=1.

    Journal: bioRxiv

    Article Title: Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies

    doi: 10.1101/2024.07.08.602472

    Figure Lengend Snippet: A: SDS PAGE of purified PspA WT and mutants (3 µg each). Purified proteins show a band at 28 kDa, except for the α0 truncated form which shows a band at 25 kDa. Asterisk: Faint dimer bands at twice the height of the monomeric protein band (56 kDa for WT and full-length PspA mutants; 50 kDa for α1-5 mutant). Marker: Bio-Rad Precision PlusProteinTM Unstained standards. B: ATPase activity of PspA. Left graph: Influence of Mg 2+ and EDTA on the ATPase activity of the WT protein. WT (orange): ATPase activity in the presence of 2 mM Mg 2+ . -Mg 2+ (green): ATPase activity in the absence of Mg 2+ . EDTA (purple): ATPase activity in the presence of 10 mM EDTA; n(WT)=36, n(-Mg 2+ and EDTA)=3. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. Middle graph: Comparison of the PspA ATPase activity with ATPase activity of E. coli PspF, n(PspA)=36, n( Eco PspF)=3. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. Right graph: Influence of ATPyS and AMPPCP on the ATPase activity of PspA. Red circle: ATPyS; Grey square: AMPPCP; n=3, error bars represent SD. C: HPLC/MS-MS of PspA directly after purification. D: HPLC/MS-MS of PspA R44K/E126Q/E179Q + ATP after 24 h incubation and extensive washing. Different color lines represent different MRM transitions. For ADP, MRM transitions are 426/134 (red) and 426/159 (light red). For ATP MRM transitions are 506/79 (dark grey) and 506/159 (light grey). The ADP fragments below the ATP peak are formed by in-source fragmentation of ATP in the ESI source. E: Bar plot of estimated nucleotide concentrations found by LC-MS/MS in PspA apo control, WT, and R44K/E126Q/E179Q rods after incubation with ATP for 24 h and extensive washing, n=1.

    Article Snippet: The ATPase activity of PspA was calculated as: with the average ATPase activity A ATPase , the phosphate release in µM ϕ[P i ] , the protein concentration in µM [Protein], and the incubation time of the reaction in hours t . To determine the ATPase activity of PspA in the presence of lipids, the ADP formed during the ATPase reaction of PspA was measured using a Promega ADP Glo Assay.

    Techniques: SDS Page, Purification, Mutagenesis, Marker, Activity Assay, Comparison, Tandem Mass Spectroscopy, Incubation, Liquid Chromatography with Mass Spectroscopy, Control

    Top. PspA rod diameter distribution for A: PspA+ATP (blue), and preformed PspA rods incubated with ATP (brown), C: PspA+ADP (red) and PspA+AMPPCP (magenta), and E: PspA+ATP (blue) and PspA+ATPyS (cyan), based on the relative occurrence of rod segments with a certain diameter. Bottom. Cross-sectional top view z -slices (left column), cross-sectional side view xy -slices (middle column) and FSC curves (threshold 0.143) of cryo-EM structures B: PspA+ATP (preformed), D: PspA+AMPPCP and F: PspA+ATPyS. G: FSC curve of PspA dL10 diameters (threshold 0.143). H: FSC curve of PspA dL10 diameters with ATP (threshold 0.143). Note that when FSC curves drop below 1, systematic peaks of high correlation occur at 1/5.3 Å corresponding to the α-helical pitch and additional low-resolution features of the all α-helical maps of PspA.

    Journal: bioRxiv

    Article Title: Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies

    doi: 10.1101/2024.07.08.602472

    Figure Lengend Snippet: Top. PspA rod diameter distribution for A: PspA+ATP (blue), and preformed PspA rods incubated with ATP (brown), C: PspA+ADP (red) and PspA+AMPPCP (magenta), and E: PspA+ATP (blue) and PspA+ATPyS (cyan), based on the relative occurrence of rod segments with a certain diameter. Bottom. Cross-sectional top view z -slices (left column), cross-sectional side view xy -slices (middle column) and FSC curves (threshold 0.143) of cryo-EM structures B: PspA+ATP (preformed), D: PspA+AMPPCP and F: PspA+ATPyS. G: FSC curve of PspA dL10 diameters (threshold 0.143). H: FSC curve of PspA dL10 diameters with ATP (threshold 0.143). Note that when FSC curves drop below 1, systematic peaks of high correlation occur at 1/5.3 Å corresponding to the α-helical pitch and additional low-resolution features of the all α-helical maps of PspA.

    Article Snippet: The ATPase activity of PspA was calculated as: with the average ATPase activity A ATPase , the phosphate release in µM ϕ[P i ] , the protein concentration in µM [Protein], and the incubation time of the reaction in hours t . To determine the ATPase activity of PspA in the presence of lipids, the ADP formed during the ATPase reaction of PspA was measured using a Promega ADP Glo Assay.

    Techniques: Incubation, Cryo-EM Sample Prep