3q4h: Difference between revisions
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< | ==Crystal structure of the Mycobacterium smegmatis EsxGH complex (MSMEG_0620-MSMEG_0621)== | ||
<StructureSection load='3q4h' size='340' side='right'caption='[[3q4h]], [[Resolution|resolution]] 2.70Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[3q4h]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Mycolicibacterium_smegmatis_MC2_155 Mycolicibacterium smegmatis MC2 155]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3Q4H OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3Q4H FirstGlance]. <br> | |||
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.7Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=MSE:SELENOMETHIONINE'>MSE</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=3q4h FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3q4h OCA], [https://pdbe.org/3q4h PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3q4h RCSB], [https://www.ebi.ac.uk/pdbsum/3q4h PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3q4h ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/ESXG_MYCS2 ESXG_MYCS2] | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes. | |||
Heterologous expression of mycobacterial Esx complexes in Escherichia coli for structural studies is facilitated by the use of maltose binding protein fusions.,Arbing MA, Chan S, Harris L, Kuo E, Zhou TT, Ahn CJ, Nguyen L, He Q, Lu J, Menchavez PT, Shin A, Holton T, Sawaya MR, Cascio D, Eisenberg D PLoS One. 2013 Nov 29;8(11):e81753. doi: 10.1371/journal.pone.0081753., eCollection 2013. PMID:24312350<ref>PMID:24312350</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
== | </div> | ||
[[ | <div class="pdbe-citations 3q4h" style="background-color:#fffaf0;"></div> | ||
[[Category: | == References == | ||
[[Category: Ahn | <references/> | ||
[[Category: Arbing | __TOC__ | ||
[[Category: Cascio | </StructureSection> | ||
[[Category: Chan | [[Category: Large Structures]] | ||
[[Category: Eisenberg | [[Category: Mycolicibacterium smegmatis MC2 155]] | ||
[[Category: Harris | [[Category: Ahn C]] | ||
[[Category: Arbing MA]] | |||
[[Category: Kuo | [[Category: Cascio D]] | ||
[[Category: Nguyen | [[Category: Chan S]] | ||
[[Category: Sawaya | [[Category: Eisenberg D]] | ||
[[Category: Shin | [[Category: Harris L]] | ||
[[Category: Kuo E]] | |||
[[Category: Zhou | [[Category: Nguyen L]] | ||
[[Category: Sawaya MR]] | |||
[[Category: Shin A]] | |||
[[Category: Zhou TT]] | |||
Latest revision as of 09:39, 27 November 2024
Crystal structure of the Mycobacterium smegmatis EsxGH complex (MSMEG_0620-MSMEG_0621)Crystal structure of the Mycobacterium smegmatis EsxGH complex (MSMEG_0620-MSMEG_0621)
Structural highlights
FunctionPublication Abstract from PubMedThe expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes. Heterologous expression of mycobacterial Esx complexes in Escherichia coli for structural studies is facilitated by the use of maltose binding protein fusions.,Arbing MA, Chan S, Harris L, Kuo E, Zhou TT, Ahn CJ, Nguyen L, He Q, Lu J, Menchavez PT, Shin A, Holton T, Sawaya MR, Cascio D, Eisenberg D PLoS One. 2013 Nov 29;8(11):e81753. doi: 10.1371/journal.pone.0081753., eCollection 2013. PMID:24312350[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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