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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.

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.

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Molecular replacement results. (a), (c) Z                   versus LLG scatter plot from coarse molecular replacement searches of inner (a) and outer (c) helical bundle ensembles. Each black square represents the top solution of one model. (b), (d) NCS*OMIT product scores resulting from coarse search for inner (b) and outer (d) helical bundle ensembles. In all panels, the top ten Z, LLG and Z*LLG scores are shown in red, the best solution from a coarse search is shown in blue, and the best solution from a fine grid search is shown in green. For clarity, the best coarse and fine search solutions are also shown as large squares in panels (a) and (c).
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fig3: Molecular replacement results. (a), (c) Z versus LLG scatter plot from coarse molecular replacement searches of inner (a) and outer (c) helical bundle ensembles. Each black square represents the top solution of one model. (b), (d) NCS*OMIT product scores resulting from coarse search for inner (b) and outer (d) helical bundle ensembles. In all panels, the top ten Z, LLG and Z*LLG scores are shown in red, the best solution from a coarse search is shown in blue, and the best solution from a fine grid search is shown in green. For clarity, the best coarse and fine search solutions are also shown as large squares in panels (a) and (c).

Mentions: The molecular replacement for the inner helical bundle (five helices) representing the inner transmembrane core of MscL was performed with a coarse set of 305 models (Table 1 ▶). The resolution range of the diffraction data was restricted to 15.0–5.0 Å in order to exclude high-resolution detail missing from our polyalanine helical models. The molecular-replacement results were represented as a scatter plot of Z versus LLG scores (Fig. 3 ▶ a). Since the inner helical bundle is a small fragment of the asymmetric unit, distinguishing the correct solution from incorrect solutions is difficult. To circumvent this problem, we assessed the models with the highest Z, LLG and Z*LLG scores (shown in red and blue in Fig. 3 ▶ a) with NCS map correlation and OMIT map correlation coefficients. The ‘best’ model, i.e. that with the largest product of NCS and OMIT map correlation, yielded the parameters r h1 = 10 Å, αh1 = 115° and βh1 = 30° (coloured blue in Figs. 3 ▶ a and 3 ▶ b). In order to obtain a higher accuracy solution for the inner helical bundle, a second finer search was performed around the top coarse solution. After repeating the process with the ‘finer’ parameters (Table 1 ▶), the resulting molecular-replacement models were again subjected to scoring by NCS and OMIT map correlation. The resulting best model yielded the parameters r h1 = 11 Å, αh1 = 120° and βh1 = 40°.


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

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

Molecular replacement results. (a), (c) Z                   versus LLG scatter plot from coarse molecular replacement searches of inner (a) and outer (c) helical bundle ensembles. Each black square represents the top solution of one model. (b), (d) NCS*OMIT product scores resulting from coarse search for inner (b) and outer (d) helical bundle ensembles. In all panels, the top ten Z, LLG and Z*LLG scores are shown in red, the best solution from a coarse search is shown in blue, and the best solution from a fine grid search is shown in green. For clarity, the best coarse and fine search solutions are also shown as large squares in panels (a) and (c).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Molecular replacement results. (a), (c) Z versus LLG scatter plot from coarse molecular replacement searches of inner (a) and outer (c) helical bundle ensembles. Each black square represents the top solution of one model. (b), (d) NCS*OMIT product scores resulting from coarse search for inner (b) and outer (d) helical bundle ensembles. In all panels, the top ten Z, LLG and Z*LLG scores are shown in red, the best solution from a coarse search is shown in blue, and the best solution from a fine grid search is shown in green. For clarity, the best coarse and fine search solutions are also shown as large squares in panels (a) and (c).
Mentions: The molecular replacement for the inner helical bundle (five helices) representing the inner transmembrane core of MscL was performed with a coarse set of 305 models (Table 1 ▶). The resolution range of the diffraction data was restricted to 15.0–5.0 Å in order to exclude high-resolution detail missing from our polyalanine helical models. The molecular-replacement results were represented as a scatter plot of Z versus LLG scores (Fig. 3 ▶ a). Since the inner helical bundle is a small fragment of the asymmetric unit, distinguishing the correct solution from incorrect solutions is difficult. To circumvent this problem, we assessed the models with the highest Z, LLG and Z*LLG scores (shown in red and blue in Fig. 3 ▶ a) with NCS map correlation and OMIT map correlation coefficients. The ‘best’ model, i.e. that with the largest product of NCS and OMIT map correlation, yielded the parameters r h1 = 10 Å, αh1 = 115° and βh1 = 30° (coloured blue in Figs. 3 ▶ a and 3 ▶ b). In order to obtain a higher accuracy solution for the inner helical bundle, a second finer search was performed around the top coarse solution. After repeating the process with the ‘finer’ parameters (Table 1 ▶), the resulting molecular-replacement models were again subjected to scoring by NCS and OMIT map correlation. The resulting best model yielded the parameters r h1 = 11 Å, αh1 = 120° and βh1 = 40°.

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