Nature 2016-02-02578BB

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A climatology of pressure jumps around the Gulf of Carpentaria

We present a climatology of pressure jumps, defined as a sharp rise in pressure of at least 0.3 hPa in three minutes, at stations around the Gulf of Carpentaria, based on one-minute data obtained from Bureau of Meteorology automatic weather stations in the region. We examine also data from one inland station, Daly Waters, which lies about 265 km west of the Gulf. Many of the pressure jumps are associated with bore-like disturbances, while others accompany seabreeze passages, or mark the onset of thunderstorm gust fronts. During the latter half of 2006, data are available at a sufficient number of stations to estimate the speed and direction of the disturbances. Bore-like disturbances tend to have two main directions of travel. The most common propagate predominantly from the northeastern sector and are associated with morning glory disturbances that originate over Cape York Peninsula. However, a number of disturbances originate south of the Gulf and subsequently propagate towards the north or northwest. These southerly morning glory disturbances are associated with the passage of cold fronts across central Australia, or are spawned within the inland trough following strong ridging across the continent. Of the 21 bore-like disturbances that were recorded at three or more stations during the four-month period August-November 2006, sixteen were from the northeastern sector and five originated south of the Gulf. One of the latter was recorded as far west as Daly Waters, where weak sea-breeze disturbances are common also, even though Daly Waters lies some 265 km inland from the Gulf. Only one northeasterly disturbance was identified at Groote Eylandt in the northwest of the Gulf

Affinity Isolation of Endogenous Saccharomyces Cerevisiae Nuclear Pore Complexes

Studying protein complexes in vitro requires the production of a relatively pure sample that maintains the full complement, native organization, and function of that complex. This can be particularly challenging to achieve for large, multi-component, membrane embedded complexes using the traditional recombinant expression and reconstitution methodologies. However, using affinity capture from native cells, suitable whole endogenous protein complexes can be isolated. Here we present a protocol for the affinity isolation of baker's yeast (S. cerevisiae) nuclear pore complexes, which are ~50 MDa assemblies made up of 552 distinct proteins and embedded in a double-membraned nuclear envelope. Producing this sample allowed us for the first time to perform analyses to characterize the mass, stoichiometry, morphology, and connectivity of this complex and to obtain its integrative structure with ~9 Å precision. We believe this methodology can be applied to other challenging protein complexes to produce similar results.This work was supported by National Institutes of Health grants U54 GM103511, R01 GM112108, P41 GM109824 and U54 DK107981.Peer reviewe

Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC

CX-MS Datasets for "Comprehensive Structure and Functional Adaptations of the Yeast Nuclear Pore Complex"

Data Files Description: NPC_XL_Identification_Inter_Crosslinked.csv: Inter-protein cross-links identified by pLink 2; NPC_XL_spectra.mgf: MS2 spectra data for the identified cross-links; NPC_XL_proteins.fasta : Protein sequences used for search.This repository contains chemical cross-linking mass spectrometry data of affinity-purified Yeast nuclear pore complexes.[Sample Processing] NPCs were immuno-purified from Mlp1 tagged S. cerevisiae strains (Kim et al., 2018). After native elution, 1.0 mM disuccinimidyl suberate (DSS) was added and the sample was incubated at 25ºC for 40 min with shaking (1,200 rpm). The reaction was quenched by adding a final concentration of 50 mM freshly prepared ammonium bicarbonate and incubating for 20 min with shaking (1,200 rpm) at 25ºC. The sample (50 µg) was then concentrated and denatured at 98ºC for 5 min in a solubilization buffer (10% solution of 1-dodecyl-3-methylimidazolium chloride (C12-mim-Cl) in 50 mM ammonium bicarbonate, pH 8.0, 100 mM DTT). After denaturation, the sample was centrifuged at 21,130 g for 10 min and the supernatant was transferred to a 100 kDa MWCO ultrafiltration unit (MRCF0R100, Microcon). The sample was quickly spun at 1,000 g for 2 min and washed twice with 50 mM ammonium bicarbonate. After alkylation (50 mM iodoacetamide), the cross-linked NPC in-filter was digested by trypsin and lysC O/N at 37ºC. After proteolysis, the sample was recovered by centrifugation and peptides were fractionated into 10-12 fractions by using a stage tip self-packed with basic C18 resins (Dr. Masch GmbH). Fractionated samples were pooled prior to LC/MS analysis. Desalted cross-link peptides were dissolved in the sample loading buffer (5% Methanol, 0.2% FA), separated with an automated nanoLC device (nLC1200, Thermo Fisher), and analyzed by an Orbitrap Q Exactive HFX (Pharma mode) mass spectrometer (Thermo Fisher) as previously described (Xiang et al., 2020; Xiang et al., 2021). Briefly, peptides were loaded onto an analytical column (C18, 1.6 μm particle size, 100 Å pore size, 75 μm × 25 cm; IonOpticks) and eluted using a 120-min liquid chromatography gradient. The flow rate was approximately 300 nl/min. The spray voltage was 1.7 kV. The QE HF-X instrument was operated in the data-dependent mode, where the top 10 most abundant ions (mass range 380 – 2,000, charge state 4 - 8) were fragmented by high-energy collisional dissociation (HCD). The target resolution was 120,000 for MS and 15,000 for tandem MS (MS/MS) analyses. The quadrupole isolation window was 1.8 Th; the maximum injection time for MS/MS was set at 200 ms.[Data processing] The raw data were searched with pLink2 (Chen et al., 2019b). An initial MS1 search window of 5 Da was allowed to cover all isotopic peaks of the cross-linked peptides. The data were automatically filtered using a mass accuracy of MS1 ≤ 10 ppm (parts per million) and MS2 ≤ 20 ppm of the theoretical monoisotopic (A0) and other isotopic masses (A+1, A+2, A+3, and A+4) as specified in the software. Other search parameters included cysteine carbamidomethyl as a fixed modification and methionine oxidation as a variable modification. A maximum of two trypsin missed-cleavage sites was allowed. The initial search results were obtained using a default 5% false discovery rate (FDR) expected by the target-decoy search strategy. Spectra were manually verified to improve data quality (Kim et al., 2018; Shi et al., 2014). Cross-linking data were analyzed and plotted with CX-Circos (http://cx-circos.net).N

Comprehensive structure and functional adaptations of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport

Integrative structure and functional anatomy of a nuclear pore complex

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Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14 MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and mRNP remodeling machinery right over the NPC's central channel, rather than on distal cytoplasmic filaments as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC