A short-term cloud forecasting method based on optical flows is used in order to increase the number of cloud free images acquired at the end of an Earth observation satellites planning process. ...Following a first validation of a satellite-based cloud forecasting method Roussel et al. 2018 for cloud fraction forecasting, it is adapted from local to global scale using parameters optimized for several climate types. This is done using a new dataset built on the basis of two nowcasting satellite application facilities products derived from meteosat second generation images, which allowed us to create a large number of test samples 0associated to six different climate types and regularly distributed throughout a whole year. Then, two test cases are performed in order to estimate the gain in terms of cloud-free acquired images proportion between the satellite-based cloud forecasting method and a more classical numerical weather prediction model, i.e., the high resolution configuration of the integrated forecasting system developed by ECMWF and Meteo-France. The first does not consider the satellites trajectory and assumes that all samples can be acquired in a single pass. The second one is realistic and imply a mission planning simulator. In both cases, the satellite-based cloud forecasting method leads to much better cloud-free images rates.
How to take your sticky proteins out safely Roussel, Guillaume; Economou, Anastassios
Proceedings of the National Academy of Sciences - PNAS,
09/2022, Letnik:
119, Številka:
39
Journal Article
Endo-inulinase is a member of glycosidase hydrolase family 32 (GH32) degrading fructans of the inulin type with an endo-cleavage mode and is an important class of industrial enzyme. In the present ...study, we report the first crystal structure of an endo-inulinase, INU2, from Aspergillus ficuum at 1.5 Å. It was solved by molecular replacement with the structure of exo-inulinase as search model. The 3D structure presents a bimodular arrangement common to other GH32 enzymes: a N-terminal 5-fold β-propeller catalytic domain with four β-sheets and a C-terminal β-sandwich domain organized in two β-sheets with five β-strands. The structural analysis and comparison with other GH32 enzymes reveal the presence of an extra pocket in the INU2 catalytic site, formed by two loops and the conserved motif W-M(I)-N-D(E)-P-N-G. This cavity would explain the endo-activity of the enzyme, the critical role of Trp40 and particularly the cleavage at the third unit of the inulin(-like) substrates. Crystal structure at 2.1 Å of INU2 complexed with fructosyl molecules, experimental digestion data and molecular modelling studies support these hypotheses.
► The first crystal structure of an endo-inulinase (from A. Ficuum) was determined. ► Its fructosyl complex points an extra pocket in the catalytic site. ► This cavity not present in exo-enzymes would explain the endo-activity of the enzyme. ► Molecular modelling supports endo-cleavage at the third fructose of the substrate.
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•The topology of SecA bound to E. coli large unilamellar vesicles was determined.•25 surface-exposed residues of a Cys-free SecA were mutated to Cys.•The polarity-sensitive ...fluorophore NBD was covalently linked to each Cys.•The disposition of SecA on the membrane in the absence of SecYEG was determined.•Conformation of SecA on the membrane is like that seen in SecA-SecYEG complexes.
The soluble cytoplasmic ATPase motor protein SecA powers protein transport across the Escherichia coli inner membrane via the SecYEG translocon. Although dimeric in solution, SecA associates monomerically with SecYEG during secretion according to several crystallographic and cryo-EM structural studies. The steps SecA follows from its dimeric cytoplasmic state to its active SecYEG monomeric state are largely unknown. We have previously shown that dimeric SecA in solution dissociates into monomers upon electrostatic binding to negatively charged lipid vesicles formed from E. coli lipids. Here we address the question of the disposition of SecA on the membrane prior to binding to membrane embedded SecYEG. We mutated to cysteine, one at a time, 25 surface-exposed residues of a Cys-free SecA. To each of these we covalently linked the polarity-sensitive fluorophore NBD whose intensity and fluorescence wavelength-shift change upon vesicle binding report on the the local membrane polarity. We established from these measurements the disposition of SecA bound to the membrane in the absence of SecYEG. Our results confirmed that SecA is anchored in the membrane interface primarily by the positive charges of the N terminus domain. But we found that a region of the nucleotide binding domain II is also important for binding. Both domains are rich in positively charged residues, consistent with electrostatic interactions playing the major role in membrane binding. Selective replacement of positively charged residues in these domains with alanine resulted in weaker binding to the membrane, which allowed us to quantitate the relative importance of the domains in stabilizing SecA on membranes. Fluorescence quenchers inside the vesicles had little effect on NBD fluorescence, indicating that SecA does not penetrate significantly across the membrane. Overall, the topology of SecA on the membrane is consistent with the conformation of SecA observed in crystallographic and cryo-EM structures of SecA-SecYEG complexes, suggesting that SecA can switch between the membrane-associated and the translocon-associated states without significant changes in conformation.
Dynamics ante portas Smit, Jochem H; Roussel, Guillaume; Economou, Anastassios
Proceedings of the National Academy of Sciences - PNAS,
07/2021, Letnik:
118, Številka:
29
Journal Article
The essential SecA motor ATPase acts in concert with the SecYEG translocon to secrete proteins into the periplasmic space of Escherichia coli. In aqueous solutions, SecA exists largely as dimers, but ...the oligomeric state on membranes is less certain. Crystallographic studies have suggested several possible solution dimeric states, but its oligomeric state when bound to membranes directly or indirectly via the translocon is controversial. We have shown using disulfide crosslinking that the principal solution dimer, corresponding to a crystallographic dimer (PDB 1M6N), binds only weakly to large unilamellar vesicles (LUV) formed from E. coli lipids. We report here that other soluble crosslinked crystallographic dimers also bind weakly, if at all, to LUV. Furthermore, using a simple glutaraldehyde crosslinking scheme, we show that SecA is always monomeric when bound to LUV formed from E. coli lipids.
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•SecA is dimeric in solution, but does it bind to lipid vesicles as a dimer?•Known crystallographic SecA dimers do not bind to E. coli-lipid vesicles.•A short-term glutaraldehyde crosslinking protocol revealed no binding of SecA dimers.•SecA-bilayer interactions cause dissociation of SecA dimers into monomers.
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