scholarly journals In situstructures of periplasmic flagella inBorreliareveals conformational changes essential for flagellar rotation

2019 ◽  
Author(s):  
Yunjie Chang ◽  
Kihwan Moon ◽  
Xiaowei Zhao ◽  
J. Norris Steven ◽  
Md A. Motaleb ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
High Speed ◽  
Electron Tomography ◽  
Proton Translocation ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Flagellar Motor ◽  
Flagellar Filament ◽  
Flagellar Rotation

SUMMARYThe bacterial flagellar motor is a molecular machine that rotates the flagellar filament at high speed. Torque is generated by the stator-rotor interaction coupled to an ion flux through the torque-generating stator. Here, we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirocheteBorrelia burgdorferi. By analysis of the motor structures of wild-type and stator mutants, we localize the torque-generating units precisely and determine three-dimensional structure of the stator and its interactions with the rotor. Our study shows that the cytoplasmic domains of the stator are packed regularly around the circumference of the flagellar C-ring. The stator-rotor interaction induces a profound conformational change in the C-ring. Analysis of the motors of a less motilemotB-D24E mutant and a non-motilemotB-D24N mutant, in which the proton translocation is reduced and blocked, respectively, provides evidence that the conformational change of the C-ring is essential for flagellar rotation.

scholarly journals Structural insights into flagellar stator–rotor interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yunjie Chang ◽  
Ki Hwan Moon ◽  
Xiaowei Zhao ◽  
Steven J Norris ◽  
MD A Motaleb ◽  
...  
Keyword(s):  
Conformational Change ◽  
High Speed ◽  
Electron Tomography ◽  
Deletion Mutants ◽  
Flagellar Motor ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Flagellar Filament ◽  
Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

scholarly journals Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

2013 ◽  
Vol 20 (1) ◽  
pp. 164-174 ◽  
Author(s):  
Gabriella Kiss ◽  
Xuemin Chen ◽  
Melinda A. Brindley ◽  
Patricia Campbell ◽  
Claudio L. Afonso ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Influenza A ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nitrilotriacetic Acid ◽  
Dimensional Structure ◽  
Enveloped Viruses ◽  
Cryo Electron Microscopy ◽  
Human Immunodeficiency Virus Virus

AbstractElectron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

scholarly journals High-resolution three-dimensional probes of biomaterials and their interfaces

2012 ◽  
Vol 370 (1963) ◽  
pp. 1337-1351 ◽  
Author(s):  
Kathryn Grandfield ◽  
Anders Palmquist ◽  
Håkan Engqvist
Keyword(s):  
High Resolution ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Controlled Drug Release ◽  
Biological Tissues ◽  
Dimensional Structure ◽  
Bone Interface ◽  
Biomaterial Interfaces ◽  
Titania Coating

Interfacial relationships between biomaterials and tissues strongly influence the success of implant materials and their long-term functionality. Owing to the inhomogeneity of biological tissues at an interface, in particular bone tissue, two-dimensional images often lack detail on the interfacial morphological complexity. Furthermore, the increasing use of nanotechnology in the design and production of biomaterials demands characterization techniques on a similar length scale. Electron tomography (ET) can meet these challenges by enabling high-resolution three-dimensional imaging of biomaterial interfaces. In this article, we review the fundamentals of ET and highlight its recent applications in probing the three-dimensional structure of bioceramics and their interfaces, with particular focus on the hydroxyapatite–bone interface, titanium dioxide–bone interface and a mesoporous titania coating for controlled drug release.

scholarly journals Biophysical and biochemical investigations of RNA catalysis in the hammerhead ribozyme

1999 ◽  
Vol 32 (3) ◽  
pp. 241-284 ◽  
Author(s):  
William G. Scott
Keyword(s):  
Metal Ions ◽  
Conformational Change ◽  
Large Scale ◽  
Enzyme Inhibitor ◽  
Hammerhead Ribozyme ◽  
Three Dimensional ◽  
Chemical Mechanism ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Hammerhead Rna

1. How do ribozymes work? 2412. The hammerhead RNA as a prototype ribozyme 2422.1 RNA enzymes 2422.2 Satellite self-cleaving RNAs 2422.3 Hammerhead RNAs and hammerhead ribozymes 2443. The chemical mechanism of hammerhead RNA self-cleavage 2463.1 Phosphodiester isomerization via an SN2(P) reaction 2473.2 The canonical role of divalent metal ions in the hammerhead ribozyme reaction 2513.3 The hammerhead ribozyme does not actually require metal ions for catalysis 2543.4 Hammerhead RNA enzyme kinetics 2574. Sequence requirements for hammerhead RNA self-cleavage 2604.1 The conserved core, mutagenesis and functional group modifications 2604.2 Ground-state vs. transition-state effects 2615. The three-dimensional structure of the hammerhead ribozyme 2625.1 Enzyme–inhibitor complexes 2625.2 Enzyme–substrate complex in the initial state 2645.3 Hammerhead ribozyme self-cleavage in the crystal 2645.4 The requirement for a conformational change 2655.5 Capture of conformational intermediates using crystallographic freeze-trapping 2665.6 The structure of a hammerhead ribozyme ‘early’ conformational intermediate 2675.7 The structure of a hammerhead ribozyme ‘later’ conformational intermediate 2685.8 Is the conformational change pH dependent? 2695.9 Isolating the structure of the cleavage product 2715.10 Evidence for and against additional large-scale conformation changes 2745.11 NMR spectroscopic studies of the hammerhead ribozyme 2786. Concluding remarks 2807. Acknowledgements 2818. References 2811. How do ribozymes work? 241The discovery that RNA can be an enzyme (Guerrier-Takada et al. 1983; Zaug & Cech, 1986) has created the fundamental question of how RNA enzymes work. Before this discovery, it was generally assumed that proteins were the only biopolymers that had sufficient complexity and chemical heterogeneity to catalyze biochemical reactions. Clearly, RNA can adopt sufficiently complex tertiary structures that make catalysis possible. How does the three- dimensional structure of an RNA endow it with catalytic activity? What structural and functional principles are unique to RNA enzymes (or ribozymes), and what principles are so fundamental that they are shared with protein enzymes?

scholarly journals A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

scholarly journals Cryo-Electron Tomography Reveals the Comparative Three-Dimensional Architecture of Prochlorococcus, a Globally Important Marine Cyanobacterium

2007 ◽  
Vol 189 (12) ◽  
pp. 4485-4493 ◽  
Author(s):  
Claire S. Ting ◽  
Chyongere Hsieh ◽  
Sesh Sundararaman ◽  
Carmen Mannella ◽  
Michael Marko
Keyword(s):  
Cell Wall ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane System ◽  
Niche Differentiation ◽  
Dimensional Structure ◽  
Comparative Genomic ◽  
Photosynthetic Membrane ◽  
Peptidoglycan Biosynthesis ◽  
Cryo Electron Tomography

ABSTRACT In an age of comparative microbial genomics, knowledge of the near-native architecture of microorganisms is essential for achieving an integrative understanding of physiology and function. We characterized and compared the three-dimensional architecture of the ecologically important cyanobacterium Prochlorococcus in a near-native state using cryo-electron tomography and found that closely related strains have diverged substantially in cellular organization and structure. By visualizing native, hydrated structures within cells, we discovered that the MED4 strain, which possesses one of the smallest genomes (1.66 Mbp) of any known photosynthetic organism, has evolved a comparatively streamlined cellular architecture. This strain possesses a smaller cell volume, an attenuated cell wall, and less extensive intracytoplasmic (photosynthetic) membrane system compared to the more deeply branched MIT9313 strain. Comparative genomic analyses indicate that differences have evolved in key structural genes, including those encoding enzymes involved in cell wall peptidoglycan biosynthesis. Although both strains possess carboxysomes that are polygonal and cluster in the central cytoplasm, the carboxysomes of MED4 are smaller. A streamlined cellular structure could be advantageous to microorganisms thriving in the low-nutrient conditions characteristic of large regions of the open ocean and thus have consequences for ecological niche differentiation. Through cryo-electron tomography we visualized, for the first time, the three-dimensional structure of the extensive network of photosynthetic lamellae within Prochlorococcus and the potential pathways for intracellular and intermembrane movement of molecules. Comparative information on the near-native structure of microorganisms is an important and necessary component of exploring microbial diversity and understanding its consequences for function and ecology.

scholarly journals The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

1998 ◽  
Vol 333 (3) ◽  
pp. 811-816 ◽  
Author(s):  
Antonio PÁRRAGA ◽  
Isabel GARCÍA-SÁEZ ◽  
Sinead B. WALSH ◽  
Timothy J. MANTLE ◽  
Miquel COLL
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Water Molecules ◽  
X Ray Diffraction ◽  
Glutathione S Transferase ◽  
X Ray ◽  
The Right

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

scholarly journals Viral Genome Conformations and Contacts across Different Lifecycle Stages

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 109
Author(s):  
Peter G. Stockley ◽  
Nikesh Patel ◽  
Emma L. Wroblewski ◽  
Andrew J. P. Scott ◽  
Carlos P. Mata ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Targets ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
Viral Genomes ◽  
Starting Point ◽  
B Virus ◽  
Infection Mechanisms ◽  
Conformational Memory

Single-stranded RNA viral genomes (gRNA) are dynamic molecules that permit packaging into virions and their subsequent extrusion during infection. For viruses with such genomes, we discovered a previously unsuspected mechanism that regulates their assembly. This regulation is the result of multiple cognate coat protein (CP)–gRNA contacts distributed across the RNA. Collectively, these interactions make the assembly highly efficient and specific. The regions of the gRNA packaging signals (PSs) driving this assembly are potential drug targets, whilst the manipulation of PS–CP contacts with nonviral RNA cargos is a route towards bespoke virus-like particles. Infectivity depends on the virions being able to transfer their gRNAs into host cells. The starting point for this transfer appears to be an encapsidated RNA with a defined three-dimensional structure, especially around the PSs. A combination of asymmetric cryo-electron microscopy structure determination and X-ray synchrotron footprinting were used to define these contacts and structures in a number of viral examples, including hepatitis B virus and enteroviruses. These tools allow us to look beyond the outer CP layer of the virion shell and to see the functional, asymmetric components that regulate viral infectivity. This revealed yet more unexpected aspects of critical infection mechanisms, such as the RNA conformational changes required for encapsidation, the details of PS–CP contacts regulating the assembly, and the conformational “memory” imposed by encapsidation.

Automation of Tilt Series Acquisition in Transmission Electron Microscopy for Applications in Biology and Materials Science

2001 ◽  
Vol 7 (S2) ◽  
pp. 88-89
Author(s):  
Ingo Daberkow ◽  
Bernhard Feja ◽  
Peter Sparlinek ◽  
Hans R. Tietz
Keyword(s):  
3D Reconstruction ◽  
Data Acquisition ◽  
San Francisco ◽  
High Speed ◽  
Materials Science ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Max Planck ◽  
Automatic Data ◽  
Tilt Series

During the last decade, computation of a three-dimensional image from a tilt series (3D reconstruction) has become a well established method, of which a variety of implementations are available. The term “electron tomography” is now generally used for this type of data acquisition and 3D reconstruction. An overview over the techniques involved is given in.With the introduction of micro-processor-controlled TEMs and cooled slow-scan CCD cameras and with the progress in performance of high-speed computers, automation of complex imaging procedures became mainly a task of developing appropriate software, using the control facilities of the microscope. in this way, automated electron tomography was realized in 1990 at the Max- Planck-Institute for Biochemistry in Martinsried, and at about the same time at the University of California in San Francisco (UCSF). New techniques for automatic focusing and alignment, developed somewhat earlier , have been integrated in these automated tomography procedures. in the following we discuss the requirements of automatic data acquisition and the present implementation for several TEMs.

Structure of Human Apolactoferrin at 2.0 Å Resolution. Refinement and Analysis of Ligand-Induced Conformational Change

1998 ◽  
Vol 54 (6) ◽  
pp. 1319-1335 ◽  
Author(s):  
Geoffrey B. Jameson ◽  
Bryan F. Anderson ◽  
Gillian E. Norris ◽  
David H. Thomas ◽  
Edward N. Baker
Keyword(s):  
Surface Area ◽  
Conformational Change ◽  
Crystal Packing ◽  
Three Dimensional ◽  
Chloride Ions ◽  
Dimensional Structure ◽  
Iron Binding ◽  
Data Set ◽  
Binding Cleft ◽  
Accessible Surface

The three-dimensional structure of a form of human apolactoferrin, in which one lobe (the N-lobe) has an open conformation and the other lobe (the C-lobe) is closed, has been refined at 2.0 Å resolution. The refinement, by restrained least-squares methods, used synchrotron radiation X-ray diffraction data combined with a lower resolution diffractometer data set. The final refined model (5346 protein atoms from residues 1–691, two Cl− ions and 363 water molecules) gives a crystallographic R factor of 0.201 (R free = 0.286) for all 51305 reflections in the resolution range 10.0–2.0 Å. The conformational change in the N-lobe, which opens up the binding cleft, involves a 54° rotation of the N2 domain relative to the N1 domain. This also results in a small reorientation of the two lobes relative to one another with a further ∼730 Å2 of surface area being buried as the N2 domain contacts the C-lobe and the inter-lobe helix. These new contacts also involve the C-terminal helix and provide a mechanism through which the conformational and iron-binding status of the N-lobe can be signalled to the C-lobe. Surface-area calculations indicate a fine balance between open and closed forms of lactoferrin, which both have essentially the same solvent-accessible surface. Chloride ions are bound in the anion-binding sites of both lobes, emphasizing the functional significance of these sites. The closed configuration of the C-lobe, attributed in part to weak stabilization by crystal packing interactions, has important implications for lactoferrin dynamics. It shows that a stable closed structure, essentially identical to that of the iron-bound form, can be formed in the absence of iron binding.

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