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1.
Mol Syst Biol ; 13(7): 936, 2017 07 25.
Article in English | MEDLINE | ID: mdl-28743795

ABSTRACT

The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known-at both the protein and complex levels-this study constitutes another step forward toward a molecular understanding of subcellular organization.


Subject(s)
Chaetomium/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Cellular Microenvironment , Cross-Linking Reagents , Cryoelectron Microscopy , Fatty Acid Synthase, Type II/chemistry , Fatty Acid Synthase, Type II/metabolism , Fatty Acid Synthase, Type II/ultrastructure , Fungal Proteins/ultrastructure , Mass Spectrometry , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Protein Interaction Mapping , Protein Interaction Maps , Proteomics , Subcellular Fractions/chemistry , Subcellular Fractions/metabolism , Systems Biology
2.
Biochem Biophys Res Commun ; 458(2): 369-74, 2015 Mar 06.
Article in English | MEDLINE | ID: mdl-25656575

ABSTRACT

Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic acid synthesis. The pathway employs a unique maoC like ß-hydroxyacyl-ACP dehydratase HadAB or HadBC heterodimer in the third step of the elongation cycle. Here we report the crystal structure of the HadAB complex determined using a Pb-SIRAS method. Crystal structure aided with enzymatic study establishes the roles of HadA as a scaffolding component and HadB as a catalytic component together indispensable for the activity. The detailed structural analysis of HadAB in combination with MD simulation endorses the spatial orientation of the central hot-dog helix and the dynamic nature of its associated loop in regulation of substrate specificities in dehydratase/hydratase family enzymes.


Subject(s)
Enoyl-CoA Hydratase/ultrastructure , Fatty Acid Synthase, Type II/chemistry , Fatty Acid Synthase, Type II/ultrastructure , Mycobacterium tuberculosis/enzymology , Amino Acid Sequence , Computer Simulation , Crystallization , Dimerization , Enoyl-CoA Hydratase/chemistry , Enoyl-CoA Hydratase/metabolism , Enzyme Activation , Fatty Acid Synthase, Type II/metabolism , Models, Chemical , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Mycobacterium tuberculosis/chemistry , Protein Binding , Protein Conformation , Protein Folding , Signal Transduction/physiology , Structure-Activity Relationship
3.
Acta Crystallogr D Biol Crystallogr ; 64(Pt 1): 76-82, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18094470

ABSTRACT

Multi-component molecular complexes are increasingly being tackled by structural biology, bringing X-ray crystallography into the purview of electron-microscopy (EM) studies. X-ray crystallography can utilize a low-resolution EM map for structure determination followed by phase extension to high resolution. Test studies have been conducted on five crystal structures of large molecular assemblies, in which EM maps are used as models for structure solution by molecular replacement (MR) using various standard MR packages such as AMoRe, MOLREP and Phaser. The results demonstrate that EM maps are viable models for molecular replacement. Possible difficulties in data analysis, such as the effects of the EM magnification error, and the effect of MR positional/rotational errors on phase extension are discussed.


Subject(s)
Crystallography, X-Ray/methods , Microscopy, Electron/methods , Fatty Acid Synthase, Type II/chemistry , Fatty Acid Synthase, Type II/ultrastructure , Models, Molecular , Protein Conformation , Software
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