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Solving structures of protein complexes by molecular replacement with Phaser.

McCoy AJ - Acta Crystallogr. D Biol. Crystallogr. (2006)

Bottom Line: Maximum-likelihood MR functions enable complex asymmetric units to be built up from individual components with a ;tree search with pruning' approach.These include cases where there are a large number of copies of the same component in the asymmetric unit or where the components of the asymmetric unit have greatly varying B factors.Two case studies are presented to illustrate how Phaser can be used to best advantage in the standard ;automated MR' mode and two case studies are used to show how to modify the automated search strategy for problematic cases.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Cambridge, Department of Haematology, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, England. ajm201@cam.ac.uk

ABSTRACT
Molecular replacement (MR) generally becomes more difficult as the number of components in the asymmetric unit requiring separate MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asymmetric units to be built up from individual components with a ;tree search with pruning' approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a number of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large number of copies of the same component in the asymmetric unit or where the components of the asymmetric unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the standard ;automated MR' mode and two case studies are used to show how to modify the automated search strategy for problematic cases.

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Catalogue of some possible contents of the unit cell for a crystal of space group P4. The contents of the asymmetric unit are as follows: top row, (a) one monomer, (b) two monomers, (c) biological homodimer, (d) two biological homodimers; middle row, (a) three biological heterodimers, (b) biological heterotetramer, (c) biological homotetramer, (d) one monomer of a biological homotetramer; bottom row, (a) one heterodimer of a biological hetero-octamer, (b) two monomers of a biological homo-octamer, (c) biological homopentamer, (d) biological heteropentamer.
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fig1: Catalogue of some possible contents of the unit cell for a crystal of space group P4. The contents of the asymmetric unit are as follows: top row, (a) one monomer, (b) two monomers, (c) biological homodimer, (d) two biological homodimers; middle row, (a) three biological heterodimers, (b) biological heterotetramer, (c) biological homotetramer, (d) one monomer of a biological homotetramer; bottom row, (a) one heterodimer of a biological hetero-octamer, (b) two monomers of a biological homo-octamer, (c) biological homopentamer, (d) biological heteropentamer.

Mentions: Although the availability of a good model is a prerequisite for MR, the quality of the target functions and search strategy are also important for success, particularly when there is an excellent model available but high symmetry, tight packing and/or multiple search components in the asymmetric unit complicate the problem. These complicating factors are often present when the target structure is a ‘biological’ protein complex (i.e. the complex is present in vivo). ‘Biological’ protein complexes can either be homo- or hetero-oligomers. The search models for hetero-oligomers are often the uncomplexed proteins, previously solved separately, and for homo-oligomers the search models are often proteins that are structurally homologous but do not form the same oligomeric association. Many combinations of crystallographic and noncrystallographic symmetry relationships between the proteins are possible. Homodimers, homotrimers, homotetramers and homohexamers may crystallize with one monomer in the asymmetric unit, with the complex generated by a crystallographic two-, three-, four- or sixfold. Hetero-oligomers or homo-oligomers in which the number of subunits is not a multiple of two, three, four or six must crystallize with at least one whole complex in the asymmetric unit. Fibres (infinite chains) must be generated by crystallographic symmetry (and may or may not also have noncrystallographic symmetry). Fig. 1 ▶ shows a schematic representation of a catalogue of possible asymmetric unit contents for a series of homo- and hetero-oligomeric protein complexes. It is important to note that the relationship between the contents of the asymmetric unit and the ‘biological’ oligomer need not be simple.


Solving structures of protein complexes by molecular replacement with Phaser.

McCoy AJ - Acta Crystallogr. D Biol. Crystallogr. (2006)

Catalogue of some possible contents of the unit cell for a crystal of space group P4. The contents of the asymmetric unit are as follows: top row, (a) one monomer, (b) two monomers, (c) biological homodimer, (d) two biological homodimers; middle row, (a) three biological heterodimers, (b) biological heterotetramer, (c) biological homotetramer, (d) one monomer of a biological homotetramer; bottom row, (a) one heterodimer of a biological hetero-octamer, (b) two monomers of a biological homo-octamer, (c) biological homopentamer, (d) biological heteropentamer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483468&req=5

fig1: Catalogue of some possible contents of the unit cell for a crystal of space group P4. The contents of the asymmetric unit are as follows: top row, (a) one monomer, (b) two monomers, (c) biological homodimer, (d) two biological homodimers; middle row, (a) three biological heterodimers, (b) biological heterotetramer, (c) biological homotetramer, (d) one monomer of a biological homotetramer; bottom row, (a) one heterodimer of a biological hetero-octamer, (b) two monomers of a biological homo-octamer, (c) biological homopentamer, (d) biological heteropentamer.
Mentions: Although the availability of a good model is a prerequisite for MR, the quality of the target functions and search strategy are also important for success, particularly when there is an excellent model available but high symmetry, tight packing and/or multiple search components in the asymmetric unit complicate the problem. These complicating factors are often present when the target structure is a ‘biological’ protein complex (i.e. the complex is present in vivo). ‘Biological’ protein complexes can either be homo- or hetero-oligomers. The search models for hetero-oligomers are often the uncomplexed proteins, previously solved separately, and for homo-oligomers the search models are often proteins that are structurally homologous but do not form the same oligomeric association. Many combinations of crystallographic and noncrystallographic symmetry relationships between the proteins are possible. Homodimers, homotrimers, homotetramers and homohexamers may crystallize with one monomer in the asymmetric unit, with the complex generated by a crystallographic two-, three-, four- or sixfold. Hetero-oligomers or homo-oligomers in which the number of subunits is not a multiple of two, three, four or six must crystallize with at least one whole complex in the asymmetric unit. Fibres (infinite chains) must be generated by crystallographic symmetry (and may or may not also have noncrystallographic symmetry). Fig. 1 ▶ shows a schematic representation of a catalogue of possible asymmetric unit contents for a series of homo- and hetero-oligomeric protein complexes. It is important to note that the relationship between the contents of the asymmetric unit and the ‘biological’ oligomer need not be simple.

Bottom Line: Maximum-likelihood MR functions enable complex asymmetric units to be built up from individual components with a ;tree search with pruning' approach.These include cases where there are a large number of copies of the same component in the asymmetric unit or where the components of the asymmetric unit have greatly varying B factors.Two case studies are presented to illustrate how Phaser can be used to best advantage in the standard ;automated MR' mode and two case studies are used to show how to modify the automated search strategy for problematic cases.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Cambridge, Department of Haematology, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, England. ajm201@cam.ac.uk

ABSTRACT
Molecular replacement (MR) generally becomes more difficult as the number of components in the asymmetric unit requiring separate MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asymmetric units to be built up from individual components with a ;tree search with pruning' approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a number of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large number of copies of the same component in the asymmetric unit or where the components of the asymmetric unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the standard ;automated MR' mode and two case studies are used to show how to modify the automated search strategy for problematic cases.

Show MeSH