Order and disorder determined from AlphaFold2 FG-containing nucleoporins: FFS and af568-HIV-1 CA assembly
FFS was performed as described by Lau et al.17. In 50 millionths of a second, the expressed GFP-Nup proteins (100 nM) and the af488 peptides were combined with the af568-HIV-1 CA assembly. Tris, pH 8, 150 mM The company is called NaCl. Fluorescence traces were recorded for 15 s per trace in 1 ms bins using a scanning stage operated at 1 μm s−1. The samples usually were repeated 10–15 times.
Owing to the length of Nup358, AlphaFold2 predictions were performed as three FG-containing sections (982–2004, 2005–3043 and 3058–3224), with their per-residue RSA-based disorder propensity calculated locally. The threshold for determining order and disorder was set by the Critical Assessment of Protein Intrinsic Disorder data.
Source: The HIV capsid mimics karyopherin engagement of FG-nucleoporins
Importin- and IBB-mCherry in pET11a : A His-fusion protein synthesis study for the preparation of nup98 and importin-anti-nuPe98
The mCherry protein was kindly provided by J. Goyette. The expression of Importin- and IBB-mCherry in pET11a was His-fusion. Cells were grown at 37 °C, protein expression was induced with 0.5 mM The growth of cells was continued overnight at 18 C. Thousands of cells were collected, and the cells were resuspended in a lysis buffer. Tris pH 7.5, 150 mM 2 mM DTT, NaMo, +cOmplete Protease Inhibitor (Roche), and lysed by sonication. The lysate was cleared by centrifugation (16,000g, 45 min, 4 °C), and the supernatant was bound to Ni-beads (Ni Sepharose High Performance, Cytiva) preincubated in purification buffer; then, imidazole was added to the protein–Ni slurry to a final concentration of 10 mM, and the mixture was incubated for 2 h at 4 °C. The beads were washed with 20 column volumes of wash buffer (purification buffer + 20 mM imidazole) and eluted in 2 ml fractions with elution buffer (purification buffer + 500 mM imidazole). Protein-containing fractions were pooled, and cleavage of the His-tag was achieved with TEV (IBB-mCherry) or SUMO (importin-β) in overnight dialysis against TEV-cleavage buffer (50 mM The SUMO-cleavage buffer (50 mM) is either Tris pH 7.5 or 1 m M DTT. The tris has a pH of 7.5. NaCl, 1 mM DTT). The flow through was collected after chellips were added to Ni-beads. A purification buffer was put in place to purify the parts of the human body. Clean protein fractions were pooled and snap frozen to be stored at −80 °C. Nup98 in pET28a was purified as described above with the following changes: the buffers used were purification buffer (6 M GuHCl, 50 mM There was a wash buffer of 20 mM imidazole and an elution buffer of 500 mM. GuHCl, 500 mM imidazole pH 8). After induction with IPTG, cells were grown further for 3 h at 30 °C before being collected. Sufficient purity of the protein was achieved with affinity chromatography; consequently, no SEC was performed. The His-tag was not removed. The 4 C temperature was where the slurry was kept for phase-separation tests.
A 63 oil immersed objective was used for the image, which was done using the Zeiss LSM 880 inverted laser scanning confocal microscope. The reaction mixes were transferred to the Ibidi chamber on a cover slide. Z-stacks were taken around the centres of the phase-separated Nup98 condensates (position with highest diameter) with sequential imaging at 488 and 568. Images were processed using ImageJ2 v.2.9.0/1.53t, MATLAB R2020a and Prism v.9.4.1.
Experimental details for Fig. 4 and Extended Data Fig. 9b–d: CLP-mCherry was assembled with a ratio of 1:100 CA-mCherry (final 0.4 µM) to CA (final 40 µM) by addition of NaCl (final 1 mM) and 15 min incubation at 37 °C. After assembly, the sample was spun down hard (18,000g, 7 min) to separate the assemblies from non-assembled monomer. The pellet was resuspended in assay buffer (50 mM Tris pH 7.5, 150 mM NaCl; resuspension volume the same as the original sample volume), followed by a slow spin of the resuspended pellet (4,000g, 5 min) to remove aggregates. After around five minutes of incubation on the coverslip, the CA-mCherry assembly was mixed with Nup98 condensates.
Experimental details for Fig. 3h,i and Extended Data Figs. 8–10: for the CPSF6P preincubation sample, CA-mCherry, CAhexamer-mCherry and CA N74Dhexamer-mCherry (65 µM final) were incubated with CPSF6 (500 µM final) for 10 min, followed by mixing with Nup98 condensates and imaging after around 5 min incubation on the coverslip. The control sample and the CPSF6 postincubation sample were incubated with MQW for 10 min, followed by mixing with Nup98 condensates and imaging after around 5 min incubation on the coverslip. For the postincubation sample, there was an added sample on the coverslip after 5, 10, 15 and 20 minutes.
The centre of the phase-separated Nup98 condensates was chosen as the location of the average radial intensity profiles. Profiles were removed from the picture. The first intensity value greater than 5 was defined as the edge of a condensate. The amount of white was subtracted to account for the point spread function. The point was defined in the radial averaged intensity graphs.
Unlabelled CLPs were prepared as described above. A two-step centrifugation protocol was performed as for confocal microscopy. Immediately prior to the plunge, equal volumes of suspension were mixed with shock-diluted condensates.
A mixture (4.5 µl) of freshly mixed Nup98 condensate and capsid with protein A-gold (10 nm) was applied onto a glow-discharged Quantifoil R2/2 copper grid (Quantifoil Micro Tools). The grid was ghatted in the front at 15 C and then plunged into liquid ethane using a Lecia Microsystem. The vitrified grids were then stored in liquid nitrogen before cryo-ET imaging.
The grids were imaged on a Talos Arctica electron microscope (Thermo Fisher Scientific) operated at 200 kV acceleration voltage. Cryo-ET data were collected with single-axis tilt on a Falcon III direct electron detector (Thermo Fisher Scientific) in linear mode at a magnification of ×28,000 with a pixel size of 5.23 Å. Tilt series were collected using the dose-symmetric scheme60 from −60 to 60° at 3° intervals using Tomography software (Thermo Fisher Scientific) with the defocus value set to −10 µm. Each tilt series had total dose ranging from 60 to 70 e/2. Before tomograms could be reconstructed, images were binned two-fold. IMOD generated three-dimensional reconstructions from the tilt series. Fiducial tracking was used to align the stack of tilted images.
There are images and graphs in this picture. There are representative data from at least three independent experiments with similar results. The images in Fig. 4a and 9 are from three independent experiments. Cryo images and graphs in Fig. 4b–h and Extended Data Fig. 9 show representative data from two independent experiments with similar results.
In vitro lysis and in situ electrophoresis of leishmania tarentolae and Nup plasmids for cell free expression
Lau et al.17 reported on the cell free expression of GFP. Briefly, Leishmania tarentolae extract was given a boost with the supplement RnaseOUT. pCellFree_03-Nup plasmids (150 ng μl−1) were then added to the expression mix, followed by incubation at 27 °C for 3 h. Following incubation, the undiluted extract was mixed 1:1 with NuPAGE LDS loading buffer (Thermo Fisher Scientific), 10 mM DTT, and the purity and degradation of the expressed Nup were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in-gel fluorescence.
They were expressed in the E. coli cells. Cultures were injected with 0.2 mM. IPTG and grown at 18 °C overnight. Cells were collected through centrifugation at 6,000g, resuspended in lysis buffer (50 mM Tris/HCl pH 8.0, 500 mM NaCl, 30 mMimidazole. Cells were lysed using a microfluidizer. Then 0.2 mM PMSF and Turbo Nuclease were added to lysate. Lysates were spun at 8,500g for 30 min. The soluble fraction was combined with Ni Sepharose 6 Fast Flow beads (Cytiva) and incubated for 1 h at 4 °C with gentle agitation. Ni-NTA binding beads were collected from samples. Beads and bound protein were washed with wash buffer (50 mM Tris/HCl pH 8.0, 300 mM NaCl, 40 mM imidazole). Beads were washed with 6 column volumes of elution buffer (50 mM Tris/HCl is acidic, 300 mM. NaCl, 250 mM imidazole). The eluted protein was dialysed into buffer (50 mM Tris/hcl has a pH 7.5 and 150 mM NaCl. The samples were stored at 80 C for later use. Purified products were analysed by an instrument known as the SDS–PAGE.
To assemble the final cross-linked compounds, they had to be mixed in a 5:1 ratio and assembled by using the following steps. The tris have a pH greater than 1,000 mM. NaCl has a 2 mM DTT against 50 mM. Tris pH 8, 1,000 mM. NaCl; and twice against 50 mM Tris pH 8, 40 mM NaCl. After being assembled, the fractions containing CAhexamer-mCherry had been pooled and frozen so that they could be kept at 80 C.
When CA-mCherry was further cleaned it was pooled and snapped frozen at a temperature of 80 C.
HIV-1 CA proteins (K158C, A204C, R18G/A204C, N57D/A204C, N74D/A204C, N77V/A204C) were expressed in Escherichia coli C41 or Rosetta2 cells and purified as previously described56. HIV-1 CA K158C was labelled with Alexa Fluor 568-C5-maleimide (AF568, Thermo Fisher Scientific, A20341) and mixed in an approximately 1:200 ratio with unlabelled HIV-1 CA A204C before assembly. The assembly of the CA lattice was done with no overnight incubation and only a 15 min incubation at 37 C. CAhexamer, CAhexamer-mCherry and CA-mCherry were purified based on previously described protocols27. Briefly, CAhexamer, CAhexamer-mCherry (pOPT) and CA-mCherry (pET21a) were expressed in E. coli C41 cells. Cell growth was continued overnight at 18 C, after it was grown at 37 C, with the help of 0.5 mM isopropyl -d-1-thiogalactopydaraside (IPTG) and a small optical density. The cells were resuspended in the lysis buffer after being collected. Tris pH 8, 50 mM NaCl, 2 mM dithiothreitol (DTT); +cOmplete EDTA-free Protease Inhibitor (Roche)) and lysed by sonication. The lysate was removed by centrifugation, and an amendment of an Ammonia sulfate was performed on the fraction. The precipitated material was pelleted at 4C after stirring for 30 min. CAhexamer was further purified by applying the pellet to the same buffer three times. The precipitatedProtein was pelleted and collected for further purification. CAhexamer-mCherry and CA-mCherry could not be purified with citric acid precipitation owing to a possible loss of mCherry fluorescence. The pellets were resuspended in buffer A and eluted in buffer B, which was a 1– 100% gradient with buffer A. The Tris has a pH 8 and 500 mM. NaCl). The parts containing proteins were pooled.
HeLa-K cells were obtained from the European Cell Culture Collection (RRID:CVCL_1922), authenticated by the manufacturer and tested negative for mycoplasma. Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, high-glucose), supplemented with fetal calf serum and antibiotics (AAS, Sigma-Aldrich) on 8-well μ-slides (IBIDI) to 70% confluence. The cells were treated with a 30 g ml1 of digitonin in the transport buffer. After 30 minutes, the cells were put into 40 nM cages with anti-Nup133 and EGFP, CA–EGFP, or a complex. Where indicated, 3 µM of a RanQ69L fragment comprising residues 1–180 and charged with GTP was also added. The samples were directly scanned with a microscope that had a 63 oil objective and 488 and 638N laser lines available to illuminate them. Such live scan directly reads the distributions of the analysed probes between bulk buffer and NPCs—although local concentration at NPCs is underestimated because of the diffraction-limited resolution.
A total of 4 µl of purified CA hexamer, CA-N21C/A22C capsid spheres and CLPs at 0.025, 0.1 and 0.25 mg ml−1, respectively, were adsorbed for 1 min on carbon-coated 300-mesh copper grids (EMS) glow-discharged for 1 min at −15 mA using a PELCO easiGlow instrument. Sample droplets were blotted with filter paper held perpendicular to the grids. The grids were then quickly washed three times in 25 μl MilliQ water droplets, followed by staining in 25 μl droplets of 2% uranyl acetate solution for 10 s and again for 1 min, with blotting between droplets. After the final staining, grids were thoroughly blotted with filter paper and allowed to air dry before imaging. Micrographs were collected on a FEI Tecnai G2 Spirit TWIN 120 kV transmission electron microscope equipped with a Gatan Ultrascan 2k × 2k CCD detector. Representative micrographs were imaged with a defocus range of 1 to 2 m and a nominal magnification of 52,000.
50 mM is contained in a high-salt buffer. The sample was taken from the tris/hcl’s concentration of 100 M IPH6, at 10–15 g/dl in room temperature. The sample was then dialysed against 50 mM A pH of 8.0 is the tris/hcl. Hexamers were isolated by size exclusion chromatography (SEC) using a Superdex 200 increase column (Cytiva) in 20 mM Tris/HCl pH 8.0, 150 mM NaCl, 100 µM IP6. Samples were stored at 80 C for later use. There was validation of the integrity of the human cell by the application of the gel electrophoresis.
Disulfide-stabilized CA hexamers were prepared as described in ref. A new one, 49. An amino-terminal His14 tag for Ni(II) chelate affinity purification and a Brachypodium distachyon (bd)SUMO tag65 were added for solubility to the CAP1A/A14C/E45C/W184A/M185A mutant. Recombinant expression was in Escherichia coli LOBSTR-RIL(DE3) (Kerafast) cells66. Liquid cultures were induced with 0.5 mM The temperature is 18 C for 16 h. Cells were resuspended in a lysis buffer. The tris/hcl ratio is 500 mM. NaCl and imidazole are both 20 mM. TCEP, 2 mM PMSF) and lysed using a high-pressure cell homogenizer (Microfluidics LM20). The lysate was cleared by centrifugation at 8,500g for 30 min. The fraction was put in a petri dish and put in a warm place for 1 h at 4 C. The beads were subsequently washed with wash buffer (50 mM Tris/hcl pH 8.0, 40 mM imidazole, 300 mM. NaCl and TCEP are both over 1 mM. Proteins were eluted from the beads by tag cleavage with 2–5 µg ml−1 of bdSenP1 in cleavage buffer (50 mM Tris/HCl pH 8.0, 150 mM For 3 h at 4 C.
Table 1 contains lists of the plasmids used for expression. The expression and purification were detailed in the references. 21,25,26,50,64).
Phase separation of FG and GLFG particles by guanidinium and tris/HCl dilution in NaCl, Tris/hcl pH 7.5 for human and yeast Nup116-FG
Partition coefficients were calculated as the raw signal in independent FG particles (in) divided by the reference areas in outside regions (out). Plots are shown for representative FG particles (with 4–7 μm diameters). Images were analysed in FIJI 2.9.0 and the exported data were further processed in GraphPad Prism 9.5.1.
The FG and GLFG stock were dissolved in 2 M guanidinium and 4 M guanidinium. Assay buffer for human Nup98-FGs and yeast Nup116-FGs was 50 mM Tris/HCl pH 7.5, 150 mM 1 mM of NaCl.
The assays were performed as previously described25,50 with minor modifications. In brief, phase separation started with a rapid reduction in the size of a 1 mM FG domain stock to 25 volumes or less of a buffer of 50 mM. The tris/hcl pH is 7.5. NaCl, 1 mM IP6), followed by another fourfold dilution in buffer containing the indicated fluorescent probes. FG and IBIDI particles were allowed to accumulate on the slides for 1 h before they could be used in confocal scans.
Source: HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor
Dissociation of ligand-loaded biosensor tips of CD1 mouse oocytes using BIO Octet RED96: Pre-incubation and image preparation
Mouse oocytes were obtained from ovaries of 9-week-old CD1 mice which were maintained in a specific pathogen-free environment according to the Federation of European Laboratory Animal Science Association guidelines and recommendations, as previously described53. After growing fully grown oocytes, they were put in homemade M2 medium with 250 M dibutyryl cyclicAMP and put under oil at 37 C. The 40 nm capsid spheres were micro injected into the nucleus of oocytes. Oocytes were imaged about 25 min after injection.
Pre-incubation of high-precision biosensor tips was done in a buffer of 20 mM. The tris/hcl is pH 8.0. NaCl, 0.1% (w/v), bovine albumin, 0.05% (v/v). Tips were then dipped into BLI buffer for 60 s. Next, biotinylated, N-terminally Avi-tagged ligands prepared in BLI buffer solution were immobilized to a thickness of 1 nm for 30–45 s. Ligand-loaded tips were then dipped into wells containing BLI buffer for 60 s, after which they were moved into a solution of HIV-1 CA hexamer in BLI buffer. Association was recorded for 180–300 s, followed by a 200–300 s dissociation step in wells containing BLI buffer. The forteBIO OctetRED96 instrument recorded all the binding sensorgrams. A reference sensor was included and used to subtract background noise. Data were normalized to the baseline step and aligned to the dissociation start point using the Octet data analysis software. Data were plotted using PRISM.