7c03: Difference between revisions
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==Crystal structure of POLArISact(T57S), genetically encoded probe for fluorescent polarization== | ==Crystal structure of POLArISact(T57S), genetically encoded probe for fluorescent polarization== | ||
<StructureSection load='7c03' size='340' side='right'caption='[[7c03]]' scene=''> | <StructureSection load='7c03' size='340' side='right'caption='[[7c03]], [[Resolution|resolution]] 2.50Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=7C03 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7C03 FirstGlance]. <br> | <table><tr><td colspan='2'>[[7c03]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Synthetic_construct Synthetic construct]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=7C03 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7C03 FirstGlance]. <br> | ||
</td></tr><tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=7c03 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7c03 OCA], [https://pdbe.org/7c03 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7c03 RCSB], [https://www.ebi.ac.uk/pdbsum/7c03 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7c03 ProSAT]</span></td></tr> | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.501Å</td></tr> | ||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=GYS:[(4Z)-2-(1-AMINO-2-HYDROXYETHYL)-4-(4-HYDROXYBENZYLIDENE)-5-OXO-4,5-DIHYDRO-1H-IMIDAZOL-1-YL]ACETIC+ACID'>GYS</scene></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=7c03 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7c03 OCA], [https://pdbe.org/7c03 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7c03 RCSB], [https://www.ebi.ac.uk/pdbsum/7c03 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7c03 ProSAT]</span></td></tr> | |||
</table> | </table> | ||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArIS(act), which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArIS(act) with FPM. In living starfish early embryos expressing POLArIS(act), we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner. | |||
POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.,Sugizaki A, Sato K, Chiba K, Saito K, Kawagishi M, Tomabechi Y, Mehta SB, Ishii H, Sakai N, Shirouzu M, Tani T, Terada S Proc Natl Acad Sci U S A. 2021 Mar 16;118(11). pii: 2019071118. doi:, 10.1073/pnas.2019071118. PMID:33674463<ref>PMID:33674463</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 7c03" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | __TOC__ | ||
</StructureSection> | </StructureSection> | ||
[[Category: Large Structures]] | [[Category: Large Structures]] | ||
[[Category: Synthetic construct]] | |||
[[Category: Sakai N]] | [[Category: Sakai N]] | ||
[[Category: Shirouzu M]] | [[Category: Shirouzu M]] | ||
[[Category: Tomabechi Y]] | [[Category: Tomabechi Y]] | ||
Revision as of 13:54, 15 November 2023
Crystal structure of POLArISact(T57S), genetically encoded probe for fluorescent polarizationCrystal structure of POLArISact(T57S), genetically encoded probe for fluorescent polarization
Structural highlights
Publication Abstract from PubMedBiomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArIS(act), which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArIS(act) with FPM. In living starfish early embryos expressing POLArIS(act), we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner. POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.,Sugizaki A, Sato K, Chiba K, Saito K, Kawagishi M, Tomabechi Y, Mehta SB, Ishii H, Sakai N, Shirouzu M, Tani T, Terada S Proc Natl Acad Sci U S A. 2021 Mar 16;118(11). pii: 2019071118. doi:, 10.1073/pnas.2019071118. PMID:33674463[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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