TY - JOUR
T1 - The axial α-helices and radial spokes in the core of the cryo-negatively stained complex flagellar filament of Pseudomonas rhodos
T2 - Recovering high-resolution details from a flexible helical assembly
AU - Cohen-Krausz, Sara
AU - Trachtenberg, Shlomo
PY - 2003/8/29
Y1 - 2003/8/29
N2 - Of the two known "complex" flagellar filaments, those of Pseudomonas are far more flexible than those of Rhizobium. Their diameter is larger and their outer three-start ridges and grooves are more prominent. Although the symmetry of both complex filaments is similar, the polymer's linear mass density and the flagellin molecular mass of the latter are lower. A recent comparison of a three-dimensional reconstruction of the filament of Pseudomonas rhodos to that of Rhizobium lupini indicates that the outer flagellin domain (D3) is missing in R.lupini. Here, we concentrate on the structure of the inner core of the filament of P.rhodos using field emission cryo-negative staining electron microscopy and a hybrid helical/single particle reconstruction technique. Averaging 158 filaments caused the density band corresponding to the radial spokes to nearly average out due to their variability and inferred flexibility. Treating the Z=0 cross-sections through the aligned individual three-dimensional density maps as images, classifying them by correspondence analysis (using a mask containing the radial spokes domain) and re-averaging the subclasses (using helical reconstruction techniques) allowed a recovery of the radial spokes and resolved the α-helices in domain D0 and the triple α-helical bundles in domain D1 at a resolution of 1/7Å-1. Although the perturbed components of the helical lattice are present along the entire filament's radius, the interior of the complex filament is similar to that of the plain one, whereas it's exterior is altered. Reconstructions of vitrified and cryo-negatively stained plain, right-handed filaments of Salmonella typhimurium SJW1655 prepared and imaged under conditions identical with those used for P.rhodos confirm the similarity of their inner cores and that the secondary structures in the interior of the flagellar filament can, under critical conditions of image recording and correction, be resolved in negative stain.
AB - Of the two known "complex" flagellar filaments, those of Pseudomonas are far more flexible than those of Rhizobium. Their diameter is larger and their outer three-start ridges and grooves are more prominent. Although the symmetry of both complex filaments is similar, the polymer's linear mass density and the flagellin molecular mass of the latter are lower. A recent comparison of a three-dimensional reconstruction of the filament of Pseudomonas rhodos to that of Rhizobium lupini indicates that the outer flagellin domain (D3) is missing in R.lupini. Here, we concentrate on the structure of the inner core of the filament of P.rhodos using field emission cryo-negative staining electron microscopy and a hybrid helical/single particle reconstruction technique. Averaging 158 filaments caused the density band corresponding to the radial spokes to nearly average out due to their variability and inferred flexibility. Treating the Z=0 cross-sections through the aligned individual three-dimensional density maps as images, classifying them by correspondence analysis (using a mask containing the radial spokes domain) and re-averaging the subclasses (using helical reconstruction techniques) allowed a recovery of the radial spokes and resolved the α-helices in domain D0 and the triple α-helical bundles in domain D1 at a resolution of 1/7Å-1. Although the perturbed components of the helical lattice are present along the entire filament's radius, the interior of the complex filament is similar to that of the plain one, whereas it's exterior is altered. Reconstructions of vitrified and cryo-negatively stained plain, right-handed filaments of Salmonella typhimurium SJW1655 prepared and imaged under conditions identical with those used for P.rhodos confirm the similarity of their inner cores and that the secondary structures in the interior of the flagellar filament can, under critical conditions of image recording and correction, be resolved in negative stain.
KW - Bacterial flagella
KW - Complex flagellar filaments
KW - Correspondence analysis
KW - Cryo-electron microscopy
KW - Flagellin
UR - http://www.scopus.com/inward/record.url?scp=0043166159&partnerID=8YFLogxK
U2 - 10.1016/S0022-2836(03)00810-6
DO - 10.1016/S0022-2836(03)00810-6
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C2 - 12927544
AN - SCOPUS:0043166159
SN - 0022-2836
VL - 331
SP - 1093
EP - 1108
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 5
ER -