Single Particle Electron Cryomicroscopy of Bacteriophage P22 Portal Protein Complexes

2008 ◽  
Vol 14 (S2) ◽  
pp. 1572-1573
Author(s):  
H Zheng ◽  
G Wisedchaisri ◽  
T Gonen
Keyword(s):  
New Mexico ◽  
Single Particle ◽  
Protein Complexes ◽  
Bacteriophage P22 ◽  
Electron Cryomicroscopy ◽  
Portal Protein

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

scholarly journals Multiple conformations facilitate PilT function in the type IV pilus

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Matthew McCallum ◽  
Samir Benlekbir ◽  
Sheryl Nguyen ◽  
Stephanie Tammam ◽  
John L. Rubinstein ◽  
...  
Keyword(s):  
Single Particle ◽  
Protein Complexes ◽  
Comprehensive Model ◽  
Type Iv Pilus ◽  
Electron Cryomicroscopy ◽  
X Ray ◽  
Functional Studies ◽  
Type Iv ◽  
Coevolution Analysis ◽  
Multiple Conformations

AbstractType IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C2 symmetry; however, most of these ATPases crystallize with either C3 or C6 symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C2,C3, and C6 conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.

scholarly journals Multiple conformations facilitate PilT function in the type IV pilus

2019 ◽  
Author(s):  
Matthew McCallum ◽  
Samir Benlekbir ◽  
Sheryl Nguyen ◽  
Stephanie Tammam ◽  
John L. Rubinstein ◽  
...  
Keyword(s):  
Single Particle ◽  
Protein Complexes ◽  
Comprehensive Model ◽  
Type Iv Pilus ◽  
Electron Cryomicroscopy ◽  
X Ray ◽  
Functional Studies ◽  
Type Iv ◽  
Evolution Analysis ◽  
Multiple Conformations

AbstractType IV pilus-like systems are protein complexes that polymerize a fibre of pilins. They are critical for virulence in many pathogens. Pilin polymerization and depolymerization are powered by motor PilT-like ATPases thought to possess C2 symmetry. However, most PilT-like ATPases crystallize with either C3 or C6 symmetry and the relevance of these conformations is unclear. Here we determined the X-ray structures of PilT in four unique conformations and used these structures to classify the conformation of available PilT-like ATPase structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT revealed condition-dependent preferences for C2,C3, and C6 conformations. The physiologic importance of these conformations was validated by co-evolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data we propose a comprehensive model of PilT function with broad implications for PilT-like ATPases.

scholarly journals Electron microscopy snapshots of single particles from single cells

2018 ◽  
Vol 294 (5) ◽  
pp. 1602-1608 ◽  
Author(s):  
Xiunan Yi ◽  
Eric J. Verbeke ◽  
Yiran Chang ◽  
Daniel J. Dickinson ◽  
David W. Taylor
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Protein Complexes ◽  
Signal To Noise Ratio ◽  
Single Cells ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
Whole Cells ◽  
Potential Applications

Cryo-electron microscopy (cryo-EM) has become an indispensable tool for structural studies of biological macromolecules. Two additional predominant methods are available for studying the architectures of multiprotein complexes: 1) single-particle analysis of purified samples and 2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, whereas the latter can capture proteins in their native state but has a low signal-to-noise ratio and yields lower-resolution structures. Here, we present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos. Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. Moreover, we investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts.

scholarly journals Single-particle tracking localization microscopy reveals nonaxonemal dynamics of intraflagellar transport proteins at the base of mammalian primary cilia

2019 ◽  
Vol 30 (7) ◽  
pp. 828-837 ◽  
Author(s):  
T. Tony Yang ◽  
Minh Nguyet Thi Tran ◽  
Weng Man Chong ◽  
Chia-En Huang ◽  
Jung-Chi Liao
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Primary Cilia ◽  
Protein Complexes ◽  
Retinal Pigment ◽  
Intraflagellar Transport ◽  
Vital Role ◽  
Single Particle Tracking ◽  
Retinal Pigment Epithelial Cells ◽  
Localization Microscopy

Primary cilia play a vital role in cellular sensing and signaling. An essential component of ciliogenesis is intraflagellar transport (IFT), which is involved in IFT protein recruitment, axonemal engagement of IFT protein complexes, and so on. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short-trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy for IFT88-mEOS4b in live human retinal pigment epithelial cells. Intriguingly, we found that mobile IFT proteins “switched gears” multiple times from the distal appendages (DAPs) to the ciliary compartment (CC), moving slowly in the DAPs, relatively fast in the proximal transition zone (TZ), slowly again in the distal TZ, and then much faster in the CC. They could travel through the space between the DAPs and the axoneme without following DAP structures. We further revealed that BBS2 and IFT88 were highly populated at the distal TZ, a potential assembly site. Together, our live-cell single-particle tracking revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliary homeostasis.

scholarly journals GraFix: sample preparation for single-particle electron cryomicroscopy

2007 ◽  
Vol 5 (1) ◽  
pp. 53-55 ◽  
Author(s):  
Berthold Kastner ◽  
Niels Fischer ◽  
Monika Mariola Golas ◽  
Bjoern Sander ◽  
Prakash Dube ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Single Particle ◽  
Electron Cryomicroscopy
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