Journal: eLife

Article Title: Ligand-coupled conformational changes in a cyclic nucleotide-gated ion channel revealed by time-resolved transition metal ion FRET

doi: 10.7554/eLife.99854

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , MBP-S361TAG -ctermSthK-TEV-TWS.pETM11 (plasmid) , Addgene , Plasmid #231098 , .

Techniques: Recombinant, Plasmid Preparation, Software

Journal: Frontiers in Plant Science

Article Title: Engineering and Modulating Functional Cyanobacterial CO 2 -Fixing Organelles

doi: 10.3389/fpls.2018.00739

Figure Lengend Snippet: Synthetic operon design and heterologous expression of synthetic β-carboxysomes in Escherichia coli . (A) Schematic representation of the molecular organization of the natural β-carboxysome operons spread in five loci within the Syn7942 genome and the synthetic β-carboxysome operons (pLFbC901 and pLFbC601) inserted in the E. coli expression vector pETM11. Locus tags are indicated. (B) Thin-section TEM images of E. coli WT and cells expressing β-carboxysome proteins using pLFbC601 and pLFbC901 plasmids. Arrows indicate β-carboxysome-like structures with polyhedral shapes observed in pLFbC601 and pLFbC901 E. coli cells. (C) In vivo carbon fixation assays of E. coli WT, pLFbC901, and pLFbC601 cells, indicating the CO -fixing activity of synthetic β-carboxysomes. A relatively higher CO -fixing activity was determined in E. coli pLFbC901 cells compared with that of E. coli pLFbC601 cells. (D) SDS-PAGE of the total cell extracts of the recombinant E. coli pLFbC901 and the E. coli WT cells. Putative β-carboxysome proteins (RbcL, RbcS, CcmM, CcmK2, CcmK4) were identified based on their molecular weights. The presence of these β-carboxysome protein blocks was further confirmed by proteomic analysis ( Supplementary Table ). The gel image was spliced based on Supplementary Figure .

Article Snippet: The operons were inserted into either the pETM11 vector digested at the EcoRI and XhoI sites or the pAM2991 vector [a gift from Susan Golden, Addgene plasmid # 40248 ( )] at the EcoRI and BamHI sites.

Techniques: Expressing, Plasmid Preparation, In Vivo, Activity Assay, SDS Page, Recombinant

Journal: Nature Communications

Article Title: Epsin and Sla2 form assemblies through phospholipid interfaces

doi: 10.1038/s41467-017-02443-x

Figure Lengend Snippet: Evolutionarily conserved features in Hip1R subfamily of ANTH domains. a Native MS was used to investigate PIP2 binding to several members of the ANTH family: ANTH Sla2 , ANTH Sla2,mut (four residues to Ala in the canonical PIP2-binding site, see Constructs) and CALM from indicated species. Signal intensities from mass spectra of at least three independent measurements were summed over all charge states, normalized to the corrected signal of the unbound protein and the average of the relative signal intensities and their standard deviations were plotted (back). After correction for unspecific clustering, relative signals of unbound and PIP2-bound ANTH domains are obtained (front). Signal for proteins with three attached PIP2 molecules observed in raw spectra disappear after correction. b Composite model of the ENTH and ANTH Sla2 complex based on the low-resolution EM structure and the high-resolution X-ray crystal structures presented here (ENTH2/PIP2, yellow, and superimposed ANTH domains of Sla2 (violet), AP180 (cyan), and CALM (pale cyan). Several elements contributing to the interface are highlighted, including the inserted α helix in the ANTH Sla2 domain, the proximate Arg 37 of ANTH Sla2 and Thr 104 from ENTH, and the location of PIP2 from the secondary site of the ENTH2/PIP2 crystal structure. c Growth defects of Sla2(1–360) strains mutated in Sla2/Hip1R ANTH-specific features. Tenfold serial dilutions of sla2Δ strains expressing Sla2(1–360) wt, Sla2(1–360) NHL with α8–α9 loop replaced by residues NHL as occurring in Yap1802, Sla2(1–360) dYL deletion of conserved Tyr 252 and Leu 253 and Sla2(1–360) R29A were incubated on SC-Ura plates at 30, 34, and 37 °C for 2 days

Article Snippet: Human CALM coding sequence has been purchased from Addgene (Plasmid #27691) and the ANTH domain was cloned in pETM-11/LIC vector for expression with an N-terminal 6xHis-tag.

Techniques: Binding Assay, Construct, Expressing, Incubation