Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module (CBM_E1) derived from sugarcane soil metagenome

doi: 10.1107/S2053230X14015520

Figure Lengend Snippet: Macromolecule cloning and expression conditions

Article Snippet: The final construct encoded full-length CBM_E1 fused to an N-terminal His tag with a thrombin protease cleavage site for tag removal. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 DNA source Sugarcane soil metagenome Forward primer † 5′-TATATAT CATATG AGCGCATCATGCGGTAGC-3′ Reverse primer † 5′-ATA GGATCC TTA CCAGTTATCGAACTTCACATT-3′ Cloning vector pJET Expression vector pET-28a Expression host Origami 2 (DE3) cells GenBank accession No. {"type":"entrez-nucleotide","attrs":{"text":"KJ917170","term_id":"684179993","term_text":"KJ917170"}} KJ917170 Open in a separate window † Restriction sites are shown in bold and the stop codon is underlined. caption a8 Macromolecule cloning and expression conditions Recombinant CBM_E1 was expressed in E. coli strain Origami 2 (DE3) (Novagen).

Techniques: Clone Assay, Expressing, Plasmid Preparation

Journal: Research Square

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.21203/rs.3.rs-3592059/v1

Figure Lengend Snippet: A) NPM1 structural features, including the secondary structure calculated from PDB:4N8M (OD), and PDB:2LLH (NTD) using DSSP, and the linear net charge per residue (LNCPR) and linear hydropathy (Hydro.) calculated using CIDER. B) p14 ARF structural features, including PSI-PRED secondary structure prediction (2°Struc.; β-strands are indicated with arrows and an α-helix with a cylinder), CIDER linear net charge per residue (LNCPR) and linear hydropathy (Hydro.), sequence conservation (Cons.) based on multi-sequence alignment using MUSCLE, and Rosetta steric zipper propensity energy (R. Energy) calculated using ZipperDB. C) CV-SANS curves for the p14 ARF -NPM1 condensed phase, which reveal the spatial organization of NPM1 (green trace), p14 ARF (blue trace) and the p14 ARF -NPM1 complex (grey trace). All curves are offset for clarity, with points shown as the average and standard deviation. Correlation peaks at ~200 Å and ~400 Å correspond to meso-scale organization of p14 ARF . D) 2D CC-DARR spectrum of [ 13 C, 15 N]-p14 ARF within the condensed phase. E) Secondary 13 C chemical shifts for [ 13 C, 15 N]-p14 ARF within the condensed phase. Assigned residues are highlighted in grey. The secondary structure prediction from panel B is shown at the top.

Article Snippet: Recombinant poly-histidine-tagged NPM1 in pET28a (+) (Novagen) were expressed in BL21 (DE3) Escherichia coli cells (Millipore Sigma, Burlington, MA, USA) grown at 37 °C in LB medium supplemented with 30 μg/ml of Kanamycin.

Techniques: Residue, Cider Assay, Sequencing, Standard Deviation

Journal: Research Square

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.21203/rs.3.rs-3592059/v1

Figure Lengend Snippet: A) 2D 1 H- 15 N TROSY-HSQC spectrum of [ 13 C, 15 N]-NPM1 within the p14 ARF -NPM1 condensed phase, displaying signals from the NPM1 IDR. B) Linear net charge per residue (LNCPR) for the NPM1 IDR. 1 H- 15 N heteronuclear NOE, R 1 and R 2 transverse relaxation profiles for NPM1 in solution (blue) and within the p14 ARF -NPM1 condensed phase (red), which show a restriction of IDR backbone motions on the ps-ns timescale. Exchange broadening rates R ex for condensed NPM1 are shown on the bottom. C ) 15 N-CPMG relaxation dispersion profiles for Ala186, A201 and T199 collected at 800 MHz, with fits to a two-state model. D) Upon phase separation with p14 ARF the NPM1 IDR exchanges slowly between multiple conformations on the μs-ms timescale. All error bars represent the standard deviations.

Article Snippet: Recombinant poly-histidine-tagged NPM1 in pET28a (+) (Novagen) were expressed in BL21 (DE3) Escherichia coli cells (Millipore Sigma, Burlington, MA, USA) grown at 37 °C in LB medium supplemented with 30 μg/ml of Kanamycin.

Techniques: Residue, Dispersion

Journal: Research Square

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.21203/rs.3.rs-3592059/v1

Figure Lengend Snippet: A) p14 ARF structural features, including PSI-PRED4.0 secondary structure (2°Struc.) prediction, CIDER linear net charge per residue (LNCPR) and CIDER linear hydropathy (Hydro.). CIDER analysis for p14 ARF ΔH1-3 is shown on the bottom. B) Confocal fluorescence micrographs of p14 ARF -NPM1 condensates (top) and p14 ARF ΔH1-3-NPM1 condensates (bottom). Scale bars = 10 μm. C) Phase diagrams for condensates shown in panel B quantified using the index of dispersion. D) CV-SANS curves for the p14 ARF ΔH1-3-NPM1 condensates; NPM1 (green trace), p14 ARF ΔH1-3 (blue trace), p14 ARF ΔH1-3-NPM1 complex (grey trace). All curves are offset for clarity, with points shown as the average and standard deviation. E) FRAP of NPM1-AF488 within condensates shows that substitution of p14 ARF hydrophobic residues to Gly/Ser spacer residues restores NPM1 mobility. F) FRAP recovery curves for p14 ARF -NPM1 and p14 ARF ΔH1-3-NPM1 condensates (n=10 for each condition, Wilcoxon rank-sum test). G) NPM1-AF488 D App values extracted from the FRAP recovery curves in panel F (n=10, Wilcoxon rank-sum test). For panels F and G, (***) p < 0.001.

Article Snippet: Recombinant poly-histidine-tagged NPM1 in pET28a (+) (Novagen) were expressed in BL21 (DE3) Escherichia coli cells (Millipore Sigma, Burlington, MA, USA) grown at 37 °C in LB medium supplemented with 30 μg/ml of Kanamycin.

Techniques: Cider Assay, Residue, Fluorescence, Dispersion, Standard Deviation

Journal: Research Square

Article Title: p14 ARF forms meso-scale assemblies upon phase separation with NPM1

doi: 10.21203/rs.3.rs-3592059/v1

Figure Lengend Snippet: A) Schematic constant-temperature and pressure phase diagram for p14 ARF -NPM1. Single phase regions are shown in white; coexistence regions are shown in gray. The curved arrow represents a concentration vector that crosses through the coexistence regions, initially sampling a liquid-like NPM1-rich phase, followed by a gel-like p14 ARF -NPM1 phase, terminating in a solid-like p14 ARF -rich phase. B) Fluorescence microscopy images of live B11 cells before and 48 hours after doxycycline induced p14 ARF -iRFP expression. Scale bars = 2 μm. C) Z-score analysis of NPM1-GFP and p14 ARF -iRFP levels in DLD-1 NPM1–G cells, showing that p14 ARF and NPM1 levels are anti-correlated (two-sided Mann-Whitney U-test, n = 2272, 122, 54), (*) p < 0.05, (****) p < 0.0001. D) Representative single-cell FRAP for two cells selected from the DLD-1 population shown in C. The curves on the left are from a cell expressing a high level of nucleolar NPM1 and low level of p14 ARF . The curves on the right are from a cell expressing a low level of nucleolar NPM1 and a high level of p14 ARF . E) The D App and F) the mobility for nucleolar NPM1-GFP is reduced as nucleolar p14 ARF -iRFP levels increase (small, transparent markers) and as the duration of p14 ARF -iRFP expression is extended (large, opaque markers). These correlated reductions in dynamics are consistent with the assembly of large molecular weight p14 ARF -NPM1 complexes. For panels E and F error bars represent the standard deviation.

Article Snippet: Recombinant poly-histidine-tagged NPM1 in pET28a (+) (Novagen) were expressed in BL21 (DE3) Escherichia coli cells (Millipore Sigma, Burlington, MA, USA) grown at 37 °C in LB medium supplemented with 30 μg/ml of Kanamycin.

Techniques: Concentration Assay, Plasmid Preparation, Sampling, Fluorescence, Microscopy, Expressing, MANN-WHITNEY, Molecular Weight, Standard Deviation