Use of a combined cryo-EM and X-ray crystallography approach to reveal molecular details of bacterial pilus assembly by the chaperone/usher pathway
Section snippets
Introduction to the combined approach of X-ray crystallography and cryo-electron microscopy
Single particle cryo-electron microscopy (SPEM) is a biophysical method for visualizing the structures of macromolecular assemblies [1]. SPEM starts with purified protein complexes in solution. Individual molecules in solution at random orientation are pipetted onto an EM grid and rapidly frozen in liquid ethane to achieve buffer vitrification. Frozen samples are kept at low temperature during electron imaging in a transmission electron microscope. A large number of particle images are
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
Work in the Li laboratory is supported by NIH grants AI70285 and GM74985 and LDRD grant 06-60 at the Brookhaven National Laboratory under contract with the US Department of Energy. Work in the Thanassi laboratory is supported by NIH grants GM62987 and AI55621.
References (51)
- et al.
Molecular motors of the bacterial flagella
Curr Opin Struct Biol
(2008) - et al.
Walking the walk: how kinesin and dynein coordinate their steps
Curr Opin Cell Biol
(2009) - et al.
The structural basis of myosin V processive movement as revealed by electron cryomicroscopy
Mol Cell
(2005) - et al.
Biogenesis of E. coli Pap pili: PapH, a minor pilin subunit involved in cell anchoring and length modulation
Cell
(1987) - et al.
Exploring the 3D molecular architecture of Escherichia coli type 1 pili
J Mol Biol
(2002) A robust algorithm for the reconstruction of helical filaments using single-particle methods
Ultramicroscopy
(2000)- et al.
Structure and assembly of P-pili: a protruding hinge region used for assembly of a bacterial adhesion filament
Proc Natl Acad Sci U S A
(2006) - et al.
Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions
Mol Cell
(2006) - et al.
Crystal structure of the P pilus rod subunit PapA
PLoS Pathog
(2007) - et al.
Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: preserved folding energy drives fiber formation
Cell
(2003)
Crystal structure of chaperone protein PapD reveals an immunoglobulin fold
Nature
Mechanism of fibre assembly through the chaperone-usher pathway
EMBO Rep
Kinetic analysis of the synthesis and assembly of type 1 fimbriae of Escherichia coli
J Bacteriol
Fiber formation across the bacterial outer membrane by the chaperone/usher pathway
Cell
The usher N terminus is the initial targeting site for chaperone-subunit complexes and participates in subsequent pilus biogenesis events
J Bacteriol
Protein translocation is mediated by oligomers of the SecY complex with one SecY copy forming the channel
Cell
Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in their Native State
Ribosome dynamics: insights from atomic structure modeling into cryo-electron microscopy maps
Annu Rev Biophys Biomol Struct
In situ structure of the complete Treponema primitia flagellar motor
Nature
Assembly of the inner rod determines needle length in the type III secretion injectisome
Nature
Structural insights into the assembly of the type III secretion needle complex
Science
FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae
Proc Natl Acad Sci U S A
P pili in uropathogenic E. coli are composite fibres with distinct fibrillar adhesive tips
Nature
Molecular mechanism of P pilus termination in uropathogenic Escherichia coli
EMBO Rep
Snapshots of usher-mediated protein secretion and ordered pilus assembly
Proc Natl Acad Sci U S A
Cited by (9)
Consensus among multiple approaches as a reliability measure for flexible fitting into cryo-EM data
2013, Journal of Structural BiologyCitation Excerpt :The emergence of cryo-electron microscopy (cryo-EM) as a structure determination technique for large macromolecular assemblies is an important development in structural biology (Campbell, 2002; Saibil, 2000; Sali et al., 2003). Cryo-EM has been used to study different conformational states of biological systems, such as the ribosome (Dunkle and Cate, 2010; Frank and Agrawal, 2000; Schmeing and Ramakrishnan, 2009; Valle et al., 2003a,b), GRoEL (Falke et al., 2005; Ranson et al., 2001), RNA polymerase (Darst et al., 2002), myosin (Wendt et al., 2001) and viruses (Conway et al., 2001; Lee and Johnson, 2003), in order to gain functional insights (Frank, 2002; Henderson, 2004; Li and Thanassi, 2009; Myasnikov et al., 2009; Subramaniam and Milne, 2004). In contrast to other experimental approaches like X-ray crystallography and NMR, electron density maps obtained from cryo-EM techniques are of intermediate to low resolution (usually 6–30 Å).
The role of SurA factor in outer membrane protein transport and virulence
2010, International Journal of Medical MicrobiologySequencing of bcfC Gene of Salmonella Typhimurium Isolated from Ducks in Egypt
2020, World's Veterinary JournalSurfaceome and proteosurfaceome in parietal monoderm bacteria: Focus on protein cell-surface display
2018, Frontiers in MicrobiologyElectrostatic networks control plug stabilization in the PapC usher
2015, Molecular Membrane BiologySecond order rate constants of donor-strand exchange reveal individual amino acid residues important in determining the subunit specificity of pilus biogenesis
2011, Journal of the American Society for Mass Spectrometry