Limits...
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.

Show MeSH

Related in: MedlinePlus

(a) Anomalous difference electron-density map (red) contoured at 4σ computed for a gold derivative using the phases obtained from ab initio molecular replacement. 2F                  o − F                  c map contoured at 1σ computed from ab initio phases with omitted residues shown in green for (b) the complete transmembrane ensemble, (c) for a single transmembrane helix and (d) for the cytoplasmic domain (for clarity, the electron density is shown for only one cytoplasmic helix).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2483470&req=5

fig6: (a) Anomalous difference electron-density map (red) contoured at 4σ computed for a gold derivative using the phases obtained from ab initio molecular replacement. 2F o − F c map contoured at 1σ computed from ab initio phases with omitted residues shown in green for (b) the complete transmembrane ensemble, (c) for a single transmembrane helix and (d) for the cytoplasmic domain (for clarity, the electron density is shown for only one cytoplasmic helix).

Mentions: To further validate the correctness of the final model, we calculated an anomalous difference electron-density map of an MscL gold derivative (Chang et al., 1998 ▶) with phases derived solely from the ab initio molecular-replacement searches. The resulting map contoured at 4σ is shown in Fig. 6 ▶(a) and clearly shows the symmetrical positions of the Au atoms. Anomalous difference maps calculated with incorrect models were not symmetrical and yielded no significant peaks at the known gold positions. We have also omitted a region of the inner helix (in all five monomers) and subjected the maps from this omitted model to a prime-and-switch density-modification protocol (Terwilliger, 2000 ▶). The resulting 2F o − F c OMIT maps contoured around the helices are shown in Figs. 6 ▶(b) and 6 ▶(c).


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

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

(a) Anomalous difference electron-density map (red) contoured at 4σ computed for a gold derivative using the phases obtained from ab initio molecular replacement. 2F                  o − F                  c map contoured at 1σ computed from ab initio phases with omitted residues shown in green for (b) the complete transmembrane ensemble, (c) for a single transmembrane helix and (d) for the cytoplasmic domain (for clarity, the electron density is shown for only one cytoplasmic helix).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: (a) Anomalous difference electron-density map (red) contoured at 4σ computed for a gold derivative using the phases obtained from ab initio molecular replacement. 2F o − F c map contoured at 1σ computed from ab initio phases with omitted residues shown in green for (b) the complete transmembrane ensemble, (c) for a single transmembrane helix and (d) for the cytoplasmic domain (for clarity, the electron density is shown for only one cytoplasmic helix).
Mentions: To further validate the correctness of the final model, we calculated an anomalous difference electron-density map of an MscL gold derivative (Chang et al., 1998 ▶) with phases derived solely from the ab initio molecular-replacement searches. The resulting map contoured at 4σ is shown in Fig. 6 ▶(a) and clearly shows the symmetrical positions of the Au atoms. Anomalous difference maps calculated with incorrect models were not symmetrical and yielded no significant peaks at the known gold positions. We have also omitted a region of the inner helix (in all five monomers) and subjected the maps from this omitted model to a prime-and-switch density-modification protocol (Terwilliger, 2000 ▶). The resulting 2F o − F c OMIT maps contoured around the helices are shown in Figs. 6 ▶(b) and 6 ▶(c).

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