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.

The study of vacuolar-type ATPases by single particle electron microscopy

2014 ◽  
Vol 92 (6) ◽  
pp. 460-466 ◽  
Author(s):  
Jianhua Zhao ◽  
John L. Rubinstein
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Catalytic Mechanism ◽  
Protein Complexes ◽  
Protein Assemblies ◽  
Functional Studies ◽  
Large Structures ◽  
Negative Stain ◽  
And Function ◽  
Complex Activities

Nature’s molecular machines often work through the concerted action of many different protein subunits, which can give rise to large structures with complex activities. Vacuolar-type ATPases (V-ATPases) are membrane-embedded protein assemblies with a unique rotary catalytic mechanism. The dynamic nature and instability of V-ATPases make structural and functional studies of these enzymes challenging. Electron microscopy (EM) techniques, especially single particle electron cryomicroscopy (cryo-EM) and negative-stain EM, have provided extensive insight into the structure and function of these protein complexes. This minireview outlines what has been learned about V-ATPases using electron microscopy, highlights current challenges for their structural study, and discusses what cryo-EM will allow us to learn about these fascinating enzymes in the future.

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

Ionic homeostasis and subcellular element compartmentation

1990 ◽  
Vol 48 (2) ◽  
pp. 150-151
Author(s):  
Ann LeFurgey ◽  
Peter Ingram ◽  
J.J. Blum ◽  
M.C. Carney ◽  
L.A. Hawkey ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Ionic Homeostasis ◽  
X Ray ◽  
Functional Studies ◽  
Cell Compartments ◽  
X Ray Imaging ◽  
Resolution Electron ◽  
Multiple Cell ◽  
Role Of Zn

Subcellular compartments commonly identified and analyzed by high resolution electron probe x-ray microanalysis (EPXMA) include mitochondria, cytoplasm and endoplasmic or sarcoplasmic reticulum. These organelles and cell regions are of primary importance in regulation of cell ionic homeostasis. Correlative structural-functional studies, based on the static probe method of EPXMA combined with biochemical and electrophysiological techniques, have focused on the role of these organelles, for example, in maintaining cell calcium homeostasis or in control of excitation-contraction coupling. New methods of real time quantitative x-ray imaging permit simultaneous examination of multiple cell compartments, especially those areas for which both membrane transport properties and element content are less well defined, e.g. nuclei including euchromatin and heterochromatin, lysosomes, mucous granules, storage vacuoles, microvilli. Investigations currently in progress have examined the role of Zn-containing polyphosphate vacuoles in the metabolism of Leishmania major, the distribution of Na, K, S and other elements during anoxia in kidney cell nuclel and lysosomes; the content and distribution of S and Ca in mucous granules of cystic fibrosis (CF) nasal epithelia; the uptake of cationic probes by mltochondria in cultured heart ceils; and the junctional sarcoplasmic retlculum (JSR) in frog skeletal muscle.

Reconstruction of Chitin-Protein Complexes Using Correlation Averaging Methods: Ovipositor of the Ichneumon Fly Megarhyssa

1980 ◽  
Vol 38 ◽  
pp. 38-39
Author(s):  
L. T. Germinario ◽  
J. Blackwell ◽  
J. Frank
Keyword(s):  
Protein Complexes ◽  
Hexagonal Lattice ◽  
Uranyl Acetate ◽  
Optical Diffraction ◽  
Insect Cuticle ◽  
High Dose ◽  
Wide Angle ◽  
X Ray ◽  
Averaging Methods ◽  
Digital Correlation

This report describes the use of digital correlation and averaging methods 1,2 for the reconstruction of high dose electron micrographs of the chitin-protein complex from Megarhyssa ovipositor. Electron microscopy of uranyl acetate stained insect cuticle has demonstrated a hexagonal array of unstained chitin monofibrils, 2.4−3.0 nm in diameter, in a stained protein matrix3,4. Optical diffraction Indicated a hexagonal lattice with a = 5.1-8.3 nm3 A particularly well ordered complex is found in the ovipositor of the ichneumon fly Megarhyssa: the small angle x-ray data gives a = 7.25 nm, and the wide angle pattern shows that the protein consists of subunits arranged in a 61 helix, with an axial repeat of 3.06 nm5.

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