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Ab initio molecular-replacement phasing for symmetric helical membrane proteins.

Strop P, Brzustowicz MR, Brunger AT - Acta Crystallogr. D Biol. Crystallogr. (2007)

Bottom Line: The number of models is significantly reduced by taking advantage of geometrical and structural restraints specific to membrane proteins.The top molecular-replacement results are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and R(free) value after refinement of a polyalanine model.The method does not require high-resolution diffraction data and can be used to obtain phases for symmetrical helical membrane proteins with one or two helices per monomer.

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Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, and Stanford Synchrotron Radiation Laboratory, Stanford University, James H. Clark Center E300, Stanford, California 94305, USA.

ABSTRACT
Obtaining phases for X-ray diffraction data can be a rate-limiting step in structure determination. Taking advantage of constraints specific to membrane proteins, an ab initio molecular-replacement method has been developed for phasing X-ray diffraction data for symmetric helical membrane proteins without prior knowledge of their structure or heavy-atom derivatives. The described method is based on generating all possible orientations of idealized transmembrane helices and using each model in a molecular-replacement search. The number of models is significantly reduced by taking advantage of geometrical and structural restraints specific to membrane proteins. The top molecular-replacement results are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and R(free) value after refinement of a polyalanine model. The feasibility of this approach is illustrated by phasing the mechanosensitive channel of large conductance (MscL) with only 4 A diffraction data. No prior structural knowledge was used other than the number of transmembrane helices. The search produced the correct spatial organization and the position in the asymmetric unit of all transmembrane helices of MscL. The resulting electron-density maps were of sufficient quality to automatically build all helical segments of MscL including the cytoplasmic domain. The method does not require high-resolution diffraction data and can be used to obtain phases for symmetrical helical membrane proteins with one or two helices per monomer.

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Comparison of (a) structure and (b) R                  free statistic for the top five converging (light blue, dark blue and green) and nonconverging (black and gray) models after torsion-angle simulated-annealing refinement with a maximum-likelihood target function. The known transmembrane structure of MscL (red) is shown for reference.
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fig4: Comparison of (a) structure and (b) R free statistic for the top five converging (light blue, dark blue and green) and nonconverging (black and gray) models after torsion-angle simulated-annealing refinement with a maximum-likelihood target function. The known transmembrane structure of MscL (red) is shown for reference.

Mentions: The correct solution was further distinguished from incorrect models by rigid-body and torsion-angle simulated-annealing refinement with a maximum-likelihood target function (Adams et al., 1999 ▶). The top five models were subjected to refinement and evaluated with the R free statistic (Fig. 4 ▶ a). Three models converged to approximately the same structure with an R free of 0.46–0.48 (Fig. 4 ▶ b). The two incorrect models are thus easily distinguisheable by their higher R free values (0.52–0.54). The final model found by ab initio molecular replacement and the known structure of MscL are qualitatively in good agreement (Figs. 5 ▶ a and 5 ▶ b).


Ab initio molecular-replacement phasing for symmetric helical membrane proteins.

Strop P, Brzustowicz MR, Brunger AT - Acta Crystallogr. D Biol. Crystallogr. (2007)

Comparison of (a) structure and (b) R                  free statistic for the top five converging (light blue, dark blue and green) and nonconverging (black and gray) models after torsion-angle simulated-annealing refinement with a maximum-likelihood target function. The known transmembrane structure of MscL (red) is shown for reference.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483470&req=5

fig4: Comparison of (a) structure and (b) R free statistic for the top five converging (light blue, dark blue and green) and nonconverging (black and gray) models after torsion-angle simulated-annealing refinement with a maximum-likelihood target function. The known transmembrane structure of MscL (red) is shown for reference.
Mentions: The correct solution was further distinguished from incorrect models by rigid-body and torsion-angle simulated-annealing refinement with a maximum-likelihood target function (Adams et al., 1999 ▶). The top five models were subjected to refinement and evaluated with the R free statistic (Fig. 4 ▶ a). Three models converged to approximately the same structure with an R free of 0.46–0.48 (Fig. 4 ▶ b). The two incorrect models are thus easily distinguisheable by their higher R free values (0.52–0.54). The final model found by ab initio molecular replacement and the known structure of MscL are qualitatively in good agreement (Figs. 5 ▶ a and 5 ▶ b).

Bottom Line: The number of models is significantly reduced by taking advantage of geometrical and structural restraints specific to membrane proteins.The top molecular-replacement results are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and R(free) value after refinement of a polyalanine model.The method does not require high-resolution diffraction data and can be used to obtain phases for symmetrical helical membrane proteins with one or two helices per monomer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, and Stanford Synchrotron Radiation Laboratory, Stanford University, James H. Clark Center E300, Stanford, California 94305, USA.

ABSTRACT
Obtaining phases for X-ray diffraction data can be a rate-limiting step in structure determination. Taking advantage of constraints specific to membrane proteins, an ab initio molecular-replacement method has been developed for phasing X-ray diffraction data for symmetric helical membrane proteins without prior knowledge of their structure or heavy-atom derivatives. The described method is based on generating all possible orientations of idealized transmembrane helices and using each model in a molecular-replacement search. The number of models is significantly reduced by taking advantage of geometrical and structural restraints specific to membrane proteins. The top molecular-replacement results are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and R(free) value after refinement of a polyalanine model. The feasibility of this approach is illustrated by phasing the mechanosensitive channel of large conductance (MscL) with only 4 A diffraction data. No prior structural knowledge was used other than the number of transmembrane helices. The search produced the correct spatial organization and the position in the asymmetric unit of all transmembrane helices of MscL. The resulting electron-density maps were of sufficient quality to automatically build all helical segments of MscL including the cytoplasmic domain. The method does not require high-resolution diffraction data and can be used to obtain phases for symmetrical helical membrane proteins with one or two helices per monomer.

Show MeSH
Related in: MedlinePlus