Journal: J Mol Biol / Year: 2006 Title: The structure of a filamentous bacteriophage. Authors: Ying A Wang / Xiong Yu / Stacy Overman / Masamichi Tsuboi / George J Thomas / Edward H Egelman / Abstract: Many thin helical polymers, including bacterial pili and filamentous bacteriophage, have been seen as refractory to high-resolution studies by electron microscopy. Studies of the quaternary structure ...Many thin helical polymers, including bacterial pili and filamentous bacteriophage, have been seen as refractory to high-resolution studies by electron microscopy. Studies of the quaternary structure of such filaments have depended upon techniques such as modeling or X-ray fiber diffraction, given that direct visualization of the subunit organization has not been possible. We report the first image reconstruction of a filamentous virus, bacteriophage fd, by cryoelectron microscopy. Although these thin ( approximately 70 A in diameter) rather featureless filaments scatter weakly, we have been able to achieve a nominal resolution of approximately 8 A using an iterative helical reconstruction procedure. We show that two different conformations of the virus exist, and that in both states the subunits are packed differently than in conflicting models previously proposed on the basis of X-ray fiber diffraction or solid-state NMR studies. A significant fraction of the population of wild-type fd is either disordered or in multiple conformational states, while in the presence of the Y21M mutation, this heterogeneity is greatly reduced, consistent with previous observations. These results show that new computational approaches to helical reconstruction can greatly extend the ability to visualize heterogeneous protein polymers at a reasonably high resolution.
Helical symmetry: (Circular symmetry: 5 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 55 / Rise per n subunits: 17.4 Å / Rotation per n subunits: -34.616 °)
Details
helical polymer
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Components
#1: Protein/peptide
CoatproteinB / Major coat protein
Mass: 5244.000 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Enterobacteria phage fd (virus) / Genus: Inovirus / Species: Enterobacteria phage M13 / Production host: Escherichia coli (E. coli) / References: UniProt: P69539
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Experimental details
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Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction
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Sample preparation
Component
ID
Name
Type
Parent-ID
Details
1
Bacteriophagefd
VIRUS
0
2
CoatproteinB
1
viralcapsid
Specimen
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Vitrification
Cryogen name: ETHANE / Method: Blot for 2 seconds before plunging
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Electron microscopy imaging
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
Microscopy
Model: FEI TECNAI F20
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD / Nominal magnification: 50000 X / Nominal defocus max: 3800 nm / Nominal defocus min: 1400 nm
Specimen holder
Specimen holder type: Gatan
Image recording
Film or detector model: KODAK SO-163 FILM
Radiation
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M
Radiation wavelength
Relative weight: 1
-
Processing
EM software
Name: IHRSR / Category: 3D reconstruction
CTF correction
Details: phase flipping of entire images
3D reconstruction
Method: IHRSR / Resolution: 8 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 16367 / Nominal pixel size: 2.4 Å / Actual pixel size: 2.4 Å / Magnification calibration: TMV Details: The C-N bond distance is 0.09 A between GLN15 and ALA16, 0.12 A between ALA35 and THR36 and 0.44 A between ALA25 and TRP26 Symmetry type: HELICAL
Refinement step
Cycle: LAST
Protein
Nucleic acid
Ligand
Solvent
Total
Num. atoms
315
0
0
0
315
+
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