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The AAA(+) motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy
Lund University.
Lund University.
Lund University.
Lund University.
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2009 (English)In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 167, no 3, 227-234 p.Article in journal (Refereed) Published
Abstract [en]

Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and COT, with molecular masses of 137, 37 and 71 kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA(+) superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and COT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and COT form a chaperone-like complex, characteristic for the AAA(+) class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.

Place, publisher, year, edition, pages
2009. Vol. 167, no 3, 227-234 p.
Keyword [en]
Metallation, Catalysis, Tetrapyrrole, Vitamin B-12, Cobalt
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-27349DOI: 10.1016/j.jsb.2009.06.013ISI: 000268775700006PubMedID: 19545636OAI: oai:DiVA.org:kth-27349DiVA: diva2:376676
Note
QC 20101213Available from: 2010-12-13 Created: 2010-12-13 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Towards unbiased 3D reconstruction: in single-particle cryo-electron microscopy
Open this publication in new window or tab >>Towards unbiased 3D reconstruction: in single-particle cryo-electron microscopy
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cryo-electron microscopy of freestanding molecules (single-particles) plays a pivotal role in the difficult and pressing challenge of determining the structures of large macromolecular complexes. Molecular volumes are generated by aligning large sets of randomly oriented two-dimensional (2D) projection images in three dimensions (3D) before reconstruction is performed using tomographic techniques. The increasing popularity of the single-particle method is highly correlated with technical advances in instrumentation and computation. This thesis introduces new computational methods for 3D structure determination from electron microscopic projection images of single molecules. The algorithms have been developed to fill a gap in the single particle methodology – the lack of methods for ab initio 3D reconstruction of asymmetrical or low-symmetry molecules co-existing in different functional states. The proposed approach does not rely on a priori information about the structure or the character of the sample heterogeneity, which minimizes template dependence and makes the methods applicable to a wide range of single molecules. The presented algorithms constitute the basis of a new open source software package - SIMPLE (Single-particle IMage Processing Linux Engine). SIMPLE is an efficient and easy-to-use image processing system for semi-automated ab initio 3D reconstruction from challenging single-particle data sets (asymmetrical particles, significant degree of heterogeneity).

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. 38 p.
Series
Trita-STH : report, ISSN 1653-3836 ; 2010:7
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-27612 (URN)978-91-7415-837-3 (ISBN)
Public defence
2011-01-14, Föreläsningssalen, Novum, Hälsovägen 7, Huddinge, 10:00 (English)
Opponent
Supervisors
Note
QC 20101214Available from: 2010-12-14 Created: 2010-12-14 Last updated: 2010-12-16Bibliographically approved

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