| 1. |
- Azinas, Stavros, et al.
(författare)
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Cryo-EM uniqueness in structure determination of macromolecular complexes : A selected structural anthology
- 2023
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Ingår i: Current opinion in structural biology. - 0959-440X .- 1879-033X. ; 81
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Tidskriftsartikel (refereegranskat)abstract
- Cryogenic electron microscopy (cryo-EM) has become in the past 10 years one of the major tools for the structure determination of proteins. Nowadays, the structure prediction field is experiencing the same revolution and, using AlphaFold2, it is possible to have high-confidence atomic models for virtually any polypeptide chain, smaller than 4000 amino acids, in a simple click. Even in a scenario where all polypeptide chain folding were to be known, cryo-EM retains specific characteristics that make it a unique tool for the structure determination of macromolecular complexes. Using cryo-EM, it is possible to obtain near-atomic structures of large and flexible megacomplexes, describe conformational panoramas, and potentially develop a structural proteomic approach from fully ex vivo specimens.
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| 2. |
- Barriga, Hanna M. G., et al.
(författare)
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Coupling Lipid Nanoparticle Structure and Automated Single-Particle Composition Analysis to Design Phospholipase-Responsive Nanocarriers
- 2022
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Ingår i: Advanced Materials. - : Wiley. - 0935-9648 .- 1521-4095. ; 34:26
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Tidskriftsartikel (refereegranskat)abstract
- Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here Single Particle Automated Raman Trapping Analysis (SPARTA) is combined with small-angle X-ray and neutron scattering (SAXS/SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP-phospholipase D (PLD) interactions. This analysis demonstrates that PLD, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate is an order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme-responsive LNP solutions for stimuli-responsive delivery and diseases where PLD is dysregulated.
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| 3. |
- Gomez-Blanco, J., et al.
(författare)
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Using Scipion for stream image processing at Cryo-EM facilities
- 2018
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Ingår i: Journal of Structural Biology. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1047-8477 .- 1095-8657. ; 204:3, s. 457-463
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Tidskriftsartikel (refereegranskat)abstract
- Three dimensional electron microscopy is becoming a very data-intensive field in which vast amounts of experimental images are acquired at high speed. To manage such large-scale projects, we had previously developed a modular workflow system called Scipion (de la Rosa-Trevfn et al., 2016). We present here a major extension of Scipion that allows processing of EM images while the data is being acquired. This approach helps to detect problems at early stages, saves computing time and provides users with a detailed evaluation of the data quality before the acquisition is finished. At present, Scipion has been deployed and is in production mode in seven Cryo-EM facilities throughout the world.
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| 4. |
- Hall, Michael, 1980-, et al.
(författare)
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Protein structural analysis by cryogenic electron microscopy
- 2023. - 1
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Ingår i: Advanced methods in structural biology. - New York : Humana Press. - 9781071631461 - 9781071631478 ; , s. 439-463
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Bokkapitel (refereegranskat)abstract
- Cryogenic electron microscopy (cryo-EM) is constantly developing and growing as a major technique for structure determination of protein complexes. Here, we detail the first steps of any cryo-EM project: specimen preparation and data collection. Step by step, a list of material needed is provided and the sequence of actions to carry out is given. We hope that these protocols will be useful to all people getting started with cryo-EM.
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| 5. |
- Johansson, Henrik J., et al.
(författare)
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Extracellular nanovesicles released from the commensal yeast Malassezia sympodialis are enriched in allergens and interact with cells in human skin
- 2018
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Malassezia sympodialis is a dominant commensal fungi in the human skin mycobiome but is also associated with common skin disorders including atopic eczema (AE). M. sympodialis releases extracellular vesicles, designated MalaEx, which are carriers of small RNAs and allergens, and they can induce inflammatory cytokine responses. Here we explored how MalaEx are involved in hostmicrobe interactions by comparing protein content of MalaEx with that of the parental yeast cells, and by investigating interactions of MalaEx with cells in the skin. Cryo-electron tomography revealed a heterogeneous population of MalaEx. iTRAQ based quantitative proteomics identified in total 2439 proteins in all replicates of which 110 were enriched in MalaEx compared to the yeast cells. Among the MalaEx enriched proteins were two of the M. sympodialis allergens, Mala s 1 and s 7. Functional experiments indicated an active binding and internalization of MalaEx into human keratinocytes and monocytes, and MalaEx were found in close proximity of the nuclei using super-resolution fluorescence 3D-SIM imaging. Our results provides new insights into host-microbe interactions, supporting that MalaEx may have a role in the sensitization and maintenance of inflammation in AE by containing enriched amounts of allergens and with their ability to interact with skin cells.
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| 6. |
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| 7. |
- Kahle, Maximilian, 1986-, et al.
(författare)
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Insights into the structure-function relationship of the NorQ/NorD chaperones from Paracoccus denitrificans reveal shared principles of interacting MoxR AAA+/VWA domain proteins
- 2023
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Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 21
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Tidskriftsartikel (refereegranskat)abstract
- Background NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families.Results We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that “plugs” the NorQ ring pore to the VWA domain of NorD with a protruding “finger” inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples.Conclusions Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.
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| 8. |
- Kudva, Renuka, et al.
(författare)
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The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding
- 2018
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Ingår i: eLIFE. - 2050-084X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The E. coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Delta loop ribosomes, while two similar to 100 residue proteins normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Delta loop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.
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| 9. |
- Madru, ClEment, et al.
(författare)
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Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA
- 2020
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77 angstrom. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography, protein-protein interaction measurements, and activity assays - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Pol alpha replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication.
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| 10. |
- Maurer, Michael, et al.
(författare)
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Toxic Activation of an AAA plus Protease by the Antibacterial Drug Cyclomarin A
- 2019
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Ingår i: Cell Chemical Biology. - : Elsevier BV. - 2451-9456 .- 2451-9448. ; 26:8, s. 1169-1179
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Tidskriftsartikel (refereegranskat)abstract
- ATP-driven bacterial AAA+ proteases have been recognized as drug targets. They possess an AAA+ protein (e.g., ClpC), which threads substrate proteins into an associated peptidase (e.g., ClpP). ATPase activity and substrate selection of AAA+ proteins are regulated by adapter proteins that bind to regulatory domains, such as the N-terminal domain (NTD). The antibacterial peptide Cyclomarin A (CymA) kills Mycobacterium tuberculosis cells by binding to the NTD of ClpC. How CymA affects ClpC function is unknown. Here, we reveal the mechanism of CymA-induced toxicity. We engineered a CymA-sensitized ClpC chimera and show that CymA activates ATPase and proteolytic activities. CymA mimics adapter binding and enables autonomous protein degradation by ClpC/ClpP with relaxed substrate selectivity. We reconstitute CymA toxicity in E. coli cells expressing engineered ClpC and ClpP, demonstrating that gain of uncontrolled proteolytic activity causes cell death. This validates drug-induced overriding of AAA+ protease activity control as effective antibacterial strategy.
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