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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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Schematic of the ab initio molecular-replacement method. Dashed lines indicate optional fine grid searches.
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fig1: Schematic of the ab initio molecular-replacement method. Dashed lines indicate optional fine grid searches.

Mentions: If one can make a reasonable ‘guess’ as to the structure (for example, from a homologous protein), molecular replacement is the method of choice since no further experimental effort is required. Indeed, as the number of structures in the Protein Data Bank (PDB) increases (Berman et al., 2000 ▶; Sussman et al., 1998 ▶), molecular-replacement methods have become increasingly more popular. However, relative to soluble proteins (∼1030 folds), the number of known membrane-protein folds (∼40 folds) is very low (Berman et al., 2000 ▶; http://scop.mrc-lmb.cam.ac.uk/scop/; Tusnady et al., 2004 ▶). Although the total number of membrane-protein folds might be smaller than the number for soluble proteins, the small numbers of presently known membrane-protein folds render molecular replacement unlikely to succeed for many cases. However, membrane proteins have the advantage that their orientation is restricted in the lipid bilayer. By surveying known α-helical membrane-protein structures, it is possible to obtain constraints on helical arrangements such as the maximum helix tilt angle, helix–helix distances and helix-packing preferences (Bowie, 1997 ▶, 1999 ▶; Spencer & Rees, 2002 ▶; Strop et al., 2003 ▶). Additionally, the number of membrane-spanning helices can be often accurately predicted from the primary sequence (Cserzo et al., 1997 ▶; Krogh et al., 2001 ▶). Taking advantage of these constraints, we have developed an ab initio molecular-replacement method for phasing X-ray diffraction data for symmetric helical membrane proteins (Fig. 1 ▶). After generating an exhaustive ensemble of plausible models, each model is subjected to a molecular-replacement search. The top molecular-replacement models are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and free R value after simulated-annealing refinement. As a test case, we successfully obtained phases for the mechanosensitive channel of large conductance (MscL; Chang et al., 1998 ▶) without any prior structural knowledge other than the number of transmembrane helices.


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

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

Schematic of the ab initio molecular-replacement method. Dashed lines indicate optional fine grid searches.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Schematic of the ab initio molecular-replacement method. Dashed lines indicate optional fine grid searches.
Mentions: If one can make a reasonable ‘guess’ as to the structure (for example, from a homologous protein), molecular replacement is the method of choice since no further experimental effort is required. Indeed, as the number of structures in the Protein Data Bank (PDB) increases (Berman et al., 2000 ▶; Sussman et al., 1998 ▶), molecular-replacement methods have become increasingly more popular. However, relative to soluble proteins (∼1030 folds), the number of known membrane-protein folds (∼40 folds) is very low (Berman et al., 2000 ▶; http://scop.mrc-lmb.cam.ac.uk/scop/; Tusnady et al., 2004 ▶). Although the total number of membrane-protein folds might be smaller than the number for soluble proteins, the small numbers of presently known membrane-protein folds render molecular replacement unlikely to succeed for many cases. However, membrane proteins have the advantage that their orientation is restricted in the lipid bilayer. By surveying known α-helical membrane-protein structures, it is possible to obtain constraints on helical arrangements such as the maximum helix tilt angle, helix–helix distances and helix-packing preferences (Bowie, 1997 ▶, 1999 ▶; Spencer & Rees, 2002 ▶; Strop et al., 2003 ▶). Additionally, the number of membrane-spanning helices can be often accurately predicted from the primary sequence (Cserzo et al., 1997 ▶; Krogh et al., 2001 ▶). Taking advantage of these constraints, we have developed an ab initio molecular-replacement method for phasing X-ray diffraction data for symmetric helical membrane proteins (Fig. 1 ▶). After generating an exhaustive ensemble of plausible models, each model is subjected to a molecular-replacement search. The top molecular-replacement models are evaluated based on noncrystallographic symmetry (NCS) map correlation, OMIT map correlation and free R value after simulated-annealing refinement. As a test case, we successfully obtained phases for the mechanosensitive channel of large conductance (MscL; Chang et al., 1998 ▶) without any prior structural knowledge other than the number of transmembrane helices.

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