jena bioscience jbscreen classic  (Jena Bioscience)


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    Name:
    JBScreen Classic 4
    Description:

    Catalog Number:
    CS-104L
    Price:
    149.0
    Category:
    Crystallography
    Size:
    24 solutions
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    Structured Review

    Jena Bioscience jena bioscience jbscreen classic
    LOV-HK crystals. (A) Screen hit #1, obtained with 30% ( v/v ) MPD, 5% ( w/v ) PEG 4000, 0.1 M Hepes, pH 7.5 <t>(Jena</t> Bioscience <t>JBScreen</t> Classic 7 Solution C4). (B) Screen hit #2, obtained with 5% ( v/v ) isopropanol, 0.1 M Hepes, pH 7.5 (Jena Bioscience JBScreen Classic 9 Solution A1). (C) Screen hit #3, obtained with 10% ( w/v ) PEG 4000, 20% ( v/v ) isopropanol, 0.1 M Hepes, pH 7.5 (Jena Bioscience JBScreen Classic 3 Solution A5). (D) Improved crystals obtained after optimization of screen hit #1. (E) Mounted crystal at the PROXIMA-2A beamline, ready for diffraction.

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    Images

    1) Product Images from "Crystallization and initial X-ray diffraction analysis of the multi-domain Brucella blue light-activated histidine kinase LOV-HK in its illuminated state"

    Article Title: Crystallization and initial X-ray diffraction analysis of the multi-domain Brucella blue light-activated histidine kinase LOV-HK in its illuminated state

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2018.09.005

    LOV-HK crystals. (A) Screen hit #1, obtained with 30% ( v/v ) MPD, 5% ( w/v ) PEG 4000, 0.1 M Hepes, pH 7.5 (Jena Bioscience JBScreen Classic 7 Solution C4). (B) Screen hit #2, obtained with 5% ( v/v ) isopropanol, 0.1 M Hepes, pH 7.5 (Jena Bioscience JBScreen Classic 9 Solution A1). (C) Screen hit #3, obtained with 10% ( w/v ) PEG 4000, 20% ( v/v ) isopropanol, 0.1 M Hepes, pH 7.5 (Jena Bioscience JBScreen Classic 3 Solution A5). (D) Improved crystals obtained after optimization of screen hit #1. (E) Mounted crystal at the PROXIMA-2A beamline, ready for diffraction.
    Figure Legend Snippet: LOV-HK crystals. (A) Screen hit #1, obtained with 30% ( v/v ) MPD, 5% ( w/v ) PEG 4000, 0.1 M Hepes, pH 7.5 (Jena Bioscience JBScreen Classic 7 Solution C4). (B) Screen hit #2, obtained with 5% ( v/v ) isopropanol, 0.1 M Hepes, pH 7.5 (Jena Bioscience JBScreen Classic 9 Solution A1). (C) Screen hit #3, obtained with 10% ( w/v ) PEG 4000, 20% ( v/v ) isopropanol, 0.1 M Hepes, pH 7.5 (Jena Bioscience JBScreen Classic 3 Solution A5). (D) Improved crystals obtained after optimization of screen hit #1. (E) Mounted crystal at the PROXIMA-2A beamline, ready for diffraction.

    Techniques Used:

    Related Articles

    Crystallization Assay:

    Article Title: Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli
    Article Snippet: The E. coli cells were cultured in Luria–Bertani (LB) medium in the presence of kanamycin (20 µg ml−1 ) and chloramphenicol (10 µg ml−1 ) for selection until the culture reached an optimal density at 660 nm and 310 K of ∼0.60. .. Initial crystallization conditions were screened by the sitting-drop vapour-diffusion method at 277 and 293 K using commercially available screening kits, including Crystal Screen HT, Index HT, SaltRx, Crystal Screen Cryo and Crystal Screen Cryo 2 (Hampton Research, Aliso Viejo, California, USA), JBScreen Classic I + II and JBScreen Cryo (Jena BioScience, Germany), NR-LBD, Clear Strategy I and II, Heavy + Light and Pact HT (Molecular Dimensions, Altamonte Springs, Florida, USA), Wizard Screen 1 and 2 and Wizard Cryo 1 and 2 (Emerald Bio, Bainbridge Island, Washington, USA). .. Crystallization drops were prepared by mixing 1 µl protein solution and 1 µl reservoir solution and were equilibrated against 80 µl reservoir solution.

    Article Title: Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA
    Article Snippet: Escherichia coli TOP10 cells were transformed with the pASK-HR3 or pASK-HR4 constructs, grown on lysogeny broth (LB) supplemented with 100 mg ml−1 ampicillin until an OD600 of 0.7 was reached and induced overnight at 293 K with 0.2 mg ml−1 anhydrotetracycline. .. Crystallization of BabA1–460 , BabA25–460 and its complex with Nb-ER19 was first screened at 293 K by the sitting-drop vapour-diffusion method with commercial sparse-matrix crystallization screening kits [SaltRx, SaltRx 2, Grid Screen Salt HT, PEGRx 2, Index HT (Hampton Research), JBScreen Basic, JBScreen Classic, JBScreen PEG/Salt (Jena Bioscience) and PACT premier HT-96, JCSG- plus HT-96 and ProPlex HT-96 (Molecular Dimensions)] in 96-well MRC plates. .. Each crystallization drop was prepared by mixing 400 nl protein solution and 400 nl reservoir solution (a 1:1 ratio) and equilibrating against 50 µl reservoir solution.

    Article Title: Crystallization and preliminary X-ray diffraction analysis of the protease from Southampton norovirus complexed with a Michael acceptor inhibitor
    Article Snippet: Further confirmation and accurate assessment of MAPI binding was accomplished by mass spectrometry, which revealed a single major peak of molecular weight 20 045 corresponding to one molecule of protease covalently linked to one molecule of the inhibitor. .. Further screening for crystallization conditions with the enzyme–inhibitor complex at a concentration of 15 mg ml−1 yielded large rhomboid crystals in 25%( w / v ) PEG 5000 MME, 100 m M Tris–HCl pH 8.5, 200 m M lithium sulfate (Jena Bioscience JBScreen Classic 4 condition A1). ..

    Article Title: Crystallization and initial X-ray diffraction analysis of the multi-domain Brucella blue light-activated histidine kinase LOV-HK in its illuminated state
    Article Snippet: Next, the protein was centrifuged at 21,000 g for 10 min at 10 °C to remove any precipitate generated at the activation step. .. Initial crystallization trials were performed at 5.3 mg ml−1 in 96-well sitting-drop vapour-diffusion Greiner 609120 plates (Monroe, North Carolina, USA) using a Honeybee 963 robot (Digilab, Marlborough, Massachusetts, USA) and the following crystallization kits: Jena Bioscience JBScreen Classic and Pentaerythritol (Jena, Germany), and Hampton Research Crystal Screen, Crystal Screen 2, PEG/Ion, PEG/Ion 2, PEGRx 1 and PEGRx 2 (Aliso Viejo, California, USA). ..

    Article Title: Expression, purification, crystallization and preliminary crystallographic analysis of an endo-1,5-?-l-arabinanase from hyperthermophilic Thermotoga petrophila
    Article Snippet: Crystallization was performed by the sitting-drop vapour-diffusion method using a Cartesian HoneyBee 963 system (Genomic Solutions). .. 544 conditions from commercially available crystallization kits were tested, including Crystal Screens I and II (Hampton Research) and JBScreen Classic and Basic kits (Jena Bioscience). .. Typically, 0.5 µl drops of protein solution (25 m M Tris–HCl buffer pH 7.5) at a concentration of 30 mg ml−1 were mixed with an equal volume of the screening solution and equilibrated over a reservoir containing 0.3 ml of the latter solution.

    Article Title: Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Zucchini from Drosophila melanogaster
    Article Snippet: The Superdex 200 10/300 column was calibrated using a Gel Filtration Calibration Kit LMW (GE Healthcare). .. Initial crystallization screening was performed at 293 K by the sitting-drop vapour-diffusion method in a 96-well MRC Crystallization Plate (Hampton Research) using screening kits including Crystal Screen, Crystal Screen 2, PEG/Ion, Natrix, Index (Hampton Research), PACT, JCSG+ (Qiagen), MemGold (Molecular Dimensions), Wizard (Emerald BioSystems), JBScreen Classic and JBScreen Membrane (Jena Bioscience). .. Crystallization drops were prepared by mixing 100 nl protein solution and 100 nl reservoir solution using a Mosquito crystallization robot (TTP LabTech).

    Concentration Assay:

    Article Title: Crystallization and preliminary X-ray diffraction analysis of the protease from Southampton norovirus complexed with a Michael acceptor inhibitor
    Article Snippet: Further confirmation and accurate assessment of MAPI binding was accomplished by mass spectrometry, which revealed a single major peak of molecular weight 20 045 corresponding to one molecule of protease covalently linked to one molecule of the inhibitor. .. Further screening for crystallization conditions with the enzyme–inhibitor complex at a concentration of 15 mg ml−1 yielded large rhomboid crystals in 25%( w / v ) PEG 5000 MME, 100 m M Tris–HCl pH 8.5, 200 m M lithium sulfate (Jena Bioscience JBScreen Classic 4 condition A1). ..

    High Throughput Screening Assay:

    Article Title: Structural characterization of the essential cell division protein FtsE and its interaction with FtsX in Streptococcus pneumoniae
    Article Snippet: Concentration of purified FtsE was determined by absorbance at 280 nm using an extinction coefficient of ε = 15,930 M−1 cm−1 calculated by the ExPASy ProtParam program ( ). .. FtsE crystallizationCrystallization screenings were performed by high-throughput techniques in a NanoDrop robot and Innovadyne SD-2 microplates (Innovadyne Technologies Inc.), screening PACT Suite and JCSG Suite (Qiagen), JBScreen Classic 1-4 and 6 (Jena Bioscience) and Crystal Screen, Crystal Screen 2 and Index HT (Hampton Research). .. The conditions that produced crystals were optimized by sitting-drop vapor-diffusion method at 291 K by mixing 1 µL of protein solution and 1 µL of precipitant solution, equilibrated against 150 µL of precipitant solution in the reservoir chamber.

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  • 90
    Jena Bioscience propargyl maleimide
    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of <t>propargyl-maleimide</t> concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.
    Propargyl Maleimide, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Jena Bioscience nourseothricin
    Interaction of Yap1- and Pdr1/Cdr1-regulated resistance pathways. (A) TDH3-YAP1 drives elevated diamide resistance independent of PDR1 or CDR1 . Isogenic wild-type, pdr1 Δ:: loxP , and cdr1 Δ:: loxP strains containing or lacking the TDH3-YAP1 allele were grown to mid-log phase and then serially diluted on plates containing the indicated concentrations of diamide. (B) The indicated strains were grown to mid-log phase and then serially diluted on rich medium containing 5 μg/ml fluconazole (FLC). (C) Complementation of fluconazole sensitivity in a cdr1 Δ flr1 Δ double mutant strain. Isogenic wild-type or cdr1 Δ flr1 Δ mutant strains were transformed with the plasmids indicated at left, which are all low-copy-number CEN-containing vectors with <t>nourseothricin</t> resistance. Both plates contain low-level nourseothricin (50 μg/ml) to ensure retention of the plasmids. (D) Diagram of interactions relating Pdr1/Cdr1 to Yap1/Flr1 and azole resistance. Arrows indicate positive interactions.
    Nourseothricin, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Jena Bioscience 3tc tp
    Zoomed active site views of the post-catalytic hPol β <t>●DNA–(−)3TC-MP●(−)3TC-TP</t> structure. (A) (−)3TC-TP bound in the open conformation following (−)3TC-MP incorporation. The N-helix is shown as a cartoon model. The F o −F c map (3 σ ) is shown as green mesh. (B) Interactions of the bound (−)3TC-TP with palm domain residues. Hydrogen bonds (dashes with distances given in Å) are formed with the backbone nitrogen atoms of G189 and S180 and the side chains of R183 and S180. Two unique hydrogen bonds are formed between the triphosphate of (−)3TC-TP and the side chain of R149. In both panels, the B-site (M B ) metal ion, shown as a sphere, is modeled as Mn 2+ . The active site residues are represented as sticks.
    3tc Tp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Jena Bioscience l tarentolae
    Aminoglycoside effects on Leishmania and the leishmanial translation machinery. a Chemical structures of AGs used in the study. Ring numbers are indicated in red; the common 2-deoxystreptamine ring (ring II) is highlighted in yellow and the substitution patterns in derivatized compounds are given as R 1 and R 2 . b In vitro inhibition of cytosolic ribosome translation in L. <t>tarentolae</t> lysates. Each value represents the mean ± standard error of at least three independent experiments performed in duplicates. Hygromycin B (HYG, orange), Geneticin (G418, blue), Paromomycin (PAR, green), Gentamicin (GEN, pink), Neomycin (NEO, yellow), and Apramycin (APR, purple). c Inhibition of L. donovani promastigote growth by AGs. Values indicate drug concentrations inducing 50% inhibition of parasite growth (LC 50 ). Each value represents the mean ± standard error of at least three independent experiments performed in triplicates
    L Tarentolae, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Journal: Free Radical Biology & Medicine

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Figure Lengend Snippet: Optimisation of the Click-PEGylation technique. (A) Effect of protein concentration on labelling efficiency during the Click-PEG reaction. Coomassie-stained mobility shift gels of purified GAPDH under untreated conditions reacted by Click-PEG red , comparing starting concentrations of 1 and 0.1 mg protein/mL. Profile plots of the ‘+ catalyst’ lanes were performed in FIJI. (B) Effect of propargyl-maleimide concentration on Click-PEG labelling. Coomassie-stained mobility shift gels of purified GAPDH reacted by Click-PEG red comparing 5 and 50 mM propargyl-maleimide, under untreated, reduced (10 mM TCEP) and oxidised (1 mM diamide) conditions. * indicates a higher molecular weight band. (C) Quantification of GAPDH thiol redox state distribution from (B). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means ±range of n=2 independent experiments. (D) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Coomassie-stained gel of purified GAPDH under reduced conditions (10 mM TCEP) reacted by Click-PEG red , comparing incubation times of 10, 30 and 120 min. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (E) Quantification of GAPDH thiol redox state distribution from (D). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments. (F) Effect of propargyl-maleimide incubation time on Click-PEG labelling. Time course as for (D), except that GAPDH redox state was assessed by Western blotting. Profile plots of the ‘–/+ catalyst’ lanes were performed in FIJI. (G) Quantification of GAPDH thiol redox state distribution from (F). Band densitometry was performed in FIJI and expressed as a % of total band intensity per lane. Data are means±range of n=2 independent experiments.

    Article Snippet: Samples were then reacted with 5 mM propargyl-maleimide at 37 °C for 30 min with agitation (1400 rpm), after which excess propargyl-maleimide was removed by passing samples through a spin column.

    Techniques: Protein Concentration, Staining, Mobility Shift, Purification, Concentration Assay, Molecular Weight, Incubation, Western Blot

    Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Journal: Free Radical Biology & Medicine

    Article Title: Click-PEGylation – A mobility shift approach to assess the redox state of cysteines in candidate proteins

    doi: 10.1016/j.freeradbiomed.2017.03.037

    Figure Lengend Snippet: Click-PEGylation schemes. (A) Principle of the Click-PEGylation reaction. A reduced thiol is alkylated with propargyl-maleimide, then conjugated with an azide-PEG of high molecular weight (e.g. 5 kDa) using copper-catalysed Click chemistry. One of two possible regioisomers is depicted. (B) Click-PEG red reaction to label reduced thiols. A protein with two potentially reversibly oxidisable cysteine residues is shown. For Click-PEG red , the sample is reacted with propargyl-maleimide to label reduced cysteines, which are subsequently derivatised with azide-PEG via Click chemistry. Optionally, oxidised cysteines can then be reduced in vitro and blocked with NEM before separation by electrophoresis. (C) Click-PEG ox reaction to label oxidised thiols. Conversely, for ClickPEG ox , the sample is first reacted with NEM to block any reduced cysteine residues. Next, previously oxidised thiols are reduced in vitro, allowing their reaction with propargyl-maleimide and derivatisation with azide-PEG. Finally, samples are separated by electrophoresis to determine the resulting redox mobility shifts.

    Article Snippet: Samples were then reacted with 5 mM propargyl-maleimide at 37 °C for 30 min with agitation (1400 rpm), after which excess propargyl-maleimide was removed by passing samples through a spin column.

    Techniques: Molecular Weight, In Vitro, Electrophoresis, Blocking Assay

    Interaction of Yap1- and Pdr1/Cdr1-regulated resistance pathways. (A) TDH3-YAP1 drives elevated diamide resistance independent of PDR1 or CDR1 . Isogenic wild-type, pdr1 Δ:: loxP , and cdr1 Δ:: loxP strains containing or lacking the TDH3-YAP1 allele were grown to mid-log phase and then serially diluted on plates containing the indicated concentrations of diamide. (B) The indicated strains were grown to mid-log phase and then serially diluted on rich medium containing 5 μg/ml fluconazole (FLC). (C) Complementation of fluconazole sensitivity in a cdr1 Δ flr1 Δ double mutant strain. Isogenic wild-type or cdr1 Δ flr1 Δ mutant strains were transformed with the plasmids indicated at left, which are all low-copy-number CEN-containing vectors with nourseothricin resistance. Both plates contain low-level nourseothricin (50 μg/ml) to ensure retention of the plasmids. (D) Diagram of interactions relating Pdr1/Cdr1 to Yap1/Flr1 and azole resistance. Arrows indicate positive interactions.

    Journal: mSphere

    Article Title: Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in Candida glabrata Drug Resistance Phenotypes

    doi: 10.1128/mSphere.00099-18

    Figure Lengend Snippet: Interaction of Yap1- and Pdr1/Cdr1-regulated resistance pathways. (A) TDH3-YAP1 drives elevated diamide resistance independent of PDR1 or CDR1 . Isogenic wild-type, pdr1 Δ:: loxP , and cdr1 Δ:: loxP strains containing or lacking the TDH3-YAP1 allele were grown to mid-log phase and then serially diluted on plates containing the indicated concentrations of diamide. (B) The indicated strains were grown to mid-log phase and then serially diluted on rich medium containing 5 μg/ml fluconazole (FLC). (C) Complementation of fluconazole sensitivity in a cdr1 Δ flr1 Δ double mutant strain. Isogenic wild-type or cdr1 Δ flr1 Δ mutant strains were transformed with the plasmids indicated at left, which are all low-copy-number CEN-containing vectors with nourseothricin resistance. Both plates contain low-level nourseothricin (50 μg/ml) to ensure retention of the plasmids. (D) Diagram of interactions relating Pdr1/Cdr1 to Yap1/Flr1 and azole resistance. Arrows indicate positive interactions.

    Article Snippet: After being heat shocked, cells were grown in YPD at 30°C at 200 rpm overnight and plated on YPD agar plates supplemented with 2 mM methionine (Fisher Scientific, Chicago, IL) and 50 µg/ml of nourseothricin (Jena Bioscience, Jena, Germany).

    Techniques: Mutagenesis, Transformation Assay, Low Copy Number

    Zoomed active site views of the post-catalytic hPol β ●DNA–(−)3TC-MP●(−)3TC-TP structure. (A) (−)3TC-TP bound in the open conformation following (−)3TC-MP incorporation. The N-helix is shown as a cartoon model. The F o −F c map (3 σ ) is shown as green mesh. (B) Interactions of the bound (−)3TC-TP with palm domain residues. Hydrogen bonds (dashes with distances given in Å) are formed with the backbone nitrogen atoms of G189 and S180 and the side chains of R183 and S180. Two unique hydrogen bonds are formed between the triphosphate of (−)3TC-TP and the side chain of R149. In both panels, the B-site (M B ) metal ion, shown as a sphere, is modeled as Mn 2+ . The active site residues are represented as sticks.

    Journal: Journal of the American Chemical Society

    Article Title: Structural Insights into the Post-Chemistry Steps of Nucleotide Incorporation Catalyzed by a DNA Polymerase

    doi: 10.1021/jacs.6b11258

    Figure Lengend Snippet: Zoomed active site views of the post-catalytic hPol β ●DNA–(−)3TC-MP●(−)3TC-TP structure. (A) (−)3TC-TP bound in the open conformation following (−)3TC-MP incorporation. The N-helix is shown as a cartoon model. The F o −F c map (3 σ ) is shown as green mesh. (B) Interactions of the bound (−)3TC-TP with palm domain residues. Hydrogen bonds (dashes with distances given in Å) are formed with the backbone nitrogen atoms of G189 and S180 and the side chains of R183 and S180. Two unique hydrogen bonds are formed between the triphosphate of (−)3TC-TP and the side chain of R149. In both panels, the B-site (M B ) metal ion, shown as a sphere, is modeled as Mn 2+ . The active site residues are represented as sticks.

    Article Snippet: The l -nucleotides, (−)FTC-TP and ()3TC-TP, were obtained from Jena Bioscience.

    Techniques:

    Superposition of post-catalytic structures of hPol β with the phosphate forms of (−)3TC. Overall superposition of the hPol β ●DNA–(−)3TC-MP●PP i (cyan) and hPol β ●DNA–(−)3TC-MP●(−)3TC-TP (green) structures. Between the two structures, the DNA substrate and protein backbone overlay well, respectively, with only a slight difference in the position of the N-heilx. (B) Active site superposition of the hPol β ●DNA–(−)3TC-MP●PP i (cyan) and hPol β ●DNA–(−)3TC-MP●(−)3TC-TP (green) structures. The side chains (R183 and S180) and backbone atoms (G189 and S180) have similar interactions with PP i and the triphosphate of (−)3TC-TP. The incoming (−)3TC-TP also has an additional interaction with R149 due to a slight repositioning of the γ -phosphate of (−)3TC-TP compared to PP i .

    Journal: Journal of the American Chemical Society

    Article Title: Structural Insights into the Post-Chemistry Steps of Nucleotide Incorporation Catalyzed by a DNA Polymerase

    doi: 10.1021/jacs.6b11258

    Figure Lengend Snippet: Superposition of post-catalytic structures of hPol β with the phosphate forms of (−)3TC. Overall superposition of the hPol β ●DNA–(−)3TC-MP●PP i (cyan) and hPol β ●DNA–(−)3TC-MP●(−)3TC-TP (green) structures. Between the two structures, the DNA substrate and protein backbone overlay well, respectively, with only a slight difference in the position of the N-heilx. (B) Active site superposition of the hPol β ●DNA–(−)3TC-MP●PP i (cyan) and hPol β ●DNA–(−)3TC-MP●(−)3TC-TP (green) structures. The side chains (R183 and S180) and backbone atoms (G189 and S180) have similar interactions with PP i and the triphosphate of (−)3TC-TP. The incoming (−)3TC-TP also has an additional interaction with R149 due to a slight repositioning of the γ -phosphate of (−)3TC-TP compared to PP i .

    Article Snippet: The l -nucleotides, (−)FTC-TP and ()3TC-TP, were obtained from Jena Bioscience.

    Techniques:

    Aminoglycoside effects on Leishmania and the leishmanial translation machinery. a Chemical structures of AGs used in the study. Ring numbers are indicated in red; the common 2-deoxystreptamine ring (ring II) is highlighted in yellow and the substitution patterns in derivatized compounds are given as R 1 and R 2 . b In vitro inhibition of cytosolic ribosome translation in L. tarentolae lysates. Each value represents the mean ± standard error of at least three independent experiments performed in duplicates. Hygromycin B (HYG, orange), Geneticin (G418, blue), Paromomycin (PAR, green), Gentamicin (GEN, pink), Neomycin (NEO, yellow), and Apramycin (APR, purple). c Inhibition of L. donovani promastigote growth by AGs. Values indicate drug concentrations inducing 50% inhibition of parasite growth (LC 50 ). Each value represents the mean ± standard error of at least three independent experiments performed in triplicates

    Journal: Nature Communications

    Article Title: Atomic resolution snapshot of Leishmania ribosome inhibition by the aminoglycoside paromomycin

    doi: 10.1038/s41467-017-01664-4

    Figure Lengend Snippet: Aminoglycoside effects on Leishmania and the leishmanial translation machinery. a Chemical structures of AGs used in the study. Ring numbers are indicated in red; the common 2-deoxystreptamine ring (ring II) is highlighted in yellow and the substitution patterns in derivatized compounds are given as R 1 and R 2 . b In vitro inhibition of cytosolic ribosome translation in L. tarentolae lysates. Each value represents the mean ± standard error of at least three independent experiments performed in duplicates. Hygromycin B (HYG, orange), Geneticin (G418, blue), Paromomycin (PAR, green), Gentamicin (GEN, pink), Neomycin (NEO, yellow), and Apramycin (APR, purple). c Inhibition of L. donovani promastigote growth by AGs. Values indicate drug concentrations inducing 50% inhibition of parasite growth (LC 50 ). Each value represents the mean ± standard error of at least three independent experiments performed in triplicates

    Article Snippet: L. tarentolae is a non-pathogenic Leishmania strain that does not cause disease in human.

    Techniques: In Vitro, Inhibition