Helical structure of unidirectionally shadowed metal replicas of cyanide hydratase from Gloeocercospora sorghi
The helical filaments of the cyanide hydratase from Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally shadowed specimens using iterative real-space helical reconstruction. The average power spectrum of all selected images has three clear reflectio...
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| Published in | Journal of structural biology Vol. 161; no. 2; pp. 111 - 119 |
|---|---|
| Main Authors | , , , , , |
| Format | Journal Article |
| Language | English |
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United States
Elsevier Inc
01.02.2008
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| Abstract | The helical filaments of the cyanide hydratase from
Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally shadowed specimens using iterative real-space helical reconstruction. The average power spectrum of all selected images has three clear reflections on different layer lines. The reconstruction is complicated by the fact that three possible indexing schemes are possible and reconstructions using the starting symmetries based on each of these indexing schemes converge on three-dimensional volumes which appear plausible. Because only one side is visible in shadowed specimens, it is necessary to examine the phases from a single filament by cryo-electron microscopy in order to make an unequivocal assignment of the symmetry. Because of the novel nature of the reconstruction method used here, conventional cryo-EM methods were also used to determine a second reconstruction, allowing us to make comparisons between the two. The filament is shown to have a left-handed one-start helix with
D
1 symmetry, 5.46 dimers per turn and a pitch of 7.15
nm. The reconstruction suggests the presence of an interaction across the groove not previously seen in nitrilase helical fibres. |
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| AbstractList | The helical filaments of the cyanide hydratase from Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally shadowed specimens using iterative real-space helical reconstruction. The average power spectrum of all selected images has three clear reflections on different layer lines. The reconstruction is complicated by the fact that three possible indexing schemes are possible and reconstructions using the starting symmetries based on each of these indexing schemes converge on three-dimensional volumes which appear plausible. Because only one side is visible in shadowed specimens, it is necessary to examine the phases from a single filament by cryo-electron microscopy in order to make an unequivocal assignment of the symmetry. Because of the novel nature of the reconstruction method used here, conventional cryo-EM methods were also used to determine a second reconstruction, allowing us to make comparisons between the two. The filament is shown to have a left-handed one-start helix with D(1) symmetry, 5.46 dimers per turn and a pitch of 7.15nm. The reconstruction suggests the presence of an interaction across the groove not previously seen in nitrilase helical fibres. The helical filaments of the cyanide hydratase from Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally shadowed specimens using iterative real-space helical reconstruction. The average power spectrum of all selected images has three clear reflections on different layer lines. The reconstruction is complicated by the fact that three possible indexing schemes are possible and reconstructions using the starting symmetries based on each of these indexing schemes converge on three-dimensional volumes which appear plausible. Because only one side is visible in shadowed specimens, it is necessary to examine the phases from a single filament by cryo-electron microscopy in order to make an unequivocal assignment of the symmetry. Because of the novel nature of the reconstruction method used here, conventional cryo-EM methods were also used to determine a second reconstruction, allowing us to make comparisons between the two. The filament is shown to have a left-handed one-start helix with D 1 symmetry, 5.46 dimers per turn and a pitch of 7.15 nm. The reconstruction suggests the presence of an interaction across the groove not previously seen in nitrilase helical fibres. |
| Author | Weber, B.W. Scheffer, M.P. Hoenger, A. Benedik, M.J. Woodward, J.D. Sewell, B.T. |
| Author_xml | – sequence: 1 givenname: J.D. surname: Woodward fullname: Woodward, J.D. organization: Electron Microscope Unit, University of Cape Town, 7701 Cape Town, South Africa – sequence: 2 givenname: B.W. surname: Weber fullname: Weber, B.W. organization: Electron Microscope Unit, University of Cape Town, 7701 Cape Town, South Africa – sequence: 3 givenname: M.P. surname: Scheffer fullname: Scheffer, M.P. organization: Electron Microscope Unit, University of Cape Town, 7701 Cape Town, South Africa – sequence: 4 givenname: M.J. surname: Benedik fullname: Benedik, M.J. organization: Department of Biology, Texas A&M University, TX, USA – sequence: 5 givenname: A. surname: Hoenger fullname: Hoenger, A. organization: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, USA – sequence: 6 givenname: B.T. surname: Sewell fullname: Sewell, B.T. email: Trevor.Sewell@uct.ac.za organization: Electron Microscope Unit, University of Cape Town, 7701 Cape Town, South Africa |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/17997328$$D View this record in MEDLINE/PubMed |
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| Keywords | Midilab IHRSR Cyanide hydratase Gloeocercospora sorghi Helical reconstruction Nitrilase Shadowing |
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Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally... The helical filaments of the cyanide hydratase from Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally... |
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| SubjectTerms | Ascomycota - enzymology Cryoelectron Microscopy Cyanide hydratase Fungal Proteins - chemistry Gloeocercospora sorghi Helical reconstruction Hydro-Lyases - chemistry Hydro-Lyases - genetics IHRSR Metals - chemistry Midilab Models, Molecular Nitrilase Protein Structure, Secondary Shadowing |
| Title | Helical structure of unidirectionally shadowed metal replicas of cyanide hydratase from Gloeocercospora sorghi |
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