scholarly journals 3H0900 Large Conformational Changes in Tubulin in the GTP- and GDPStates Microtubules Observed by Cryo Electron Microscopy(Cell Biology III:Cytoskeleton & Motility,Oral Presentation)

2012 ◽  
Vol 52 (supplement) ◽  
pp. S69
Author(s):  
Hiroaki Yajima ◽  
Toshihiko Ogura ◽  
Ryo Nitta ◽  
Yasushi Okada ◽  
Chikara Sato ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Oral Presentation ◽  
Cell Biology ◽  
Cryo Electron Microscopy

scholarly journals The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Tomáš Kouba ◽  
Jiří Pospíšil ◽  
Jarmila Hnilicová ◽  
Hana Šanderová ◽  
Ivan Barvík ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Cryo Electron Microscopy ◽  
Bacterial Rna ◽  
High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

scholarly journals The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy

2015 ◽  
Vol 89 (23) ◽  
pp. 12108-12117 ◽  
Author(s):  
Jian Guan ◽  
Stephanie M. Bywaters ◽  
Sarah A. Brendle ◽  
Hyunwook Lee ◽  
Robert E. Ashley ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Human Papillomavirus ◽  
Conformational Changes ◽  
Vaccine Development ◽  
Contact Point ◽  
Human Papillomavirus 16 ◽  
Cryo Electron Microscopy ◽  
C Terminus ◽  
L1 Protein

ABSTRACTThe human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers.IMPORTANCEHuman papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.

scholarly journals Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Joseph Atherton ◽  
Irene Farabella ◽  
I-Mei Yu ◽  
Steven S Rosenfeld ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Catalytic Site ◽  
Fundamental Question ◽  
Force Generation ◽  
Atp Binding ◽  
Cellular Functions ◽  
Microtubule Binding ◽  
Cryo Electron Microscopy ◽  
Adp Release

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.

scholarly journals Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus

2016 ◽  
Vol 90 (21) ◽  
pp. 9733-9742 ◽  
Author(s):  
Lindsey J. Organtini ◽  
Hyunwook Lee ◽  
Sho Iketani ◽  
Kai Huang ◽  
Robert E. Ashley ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Receptor Binding ◽  
Conformational Changes ◽  
De Novo ◽  
Mutational Analysis ◽  
Antibody Fragment ◽  
Canine Parvovirus ◽  
Single Chain ◽  
Cryo Electron Microscopy ◽  
Resolution Structure

ABSTRACT Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. IMPORTANCE Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time.

scholarly journals Structure of the human epithelial sodium channel by cryo-electron microscopy

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sigrid Noreng ◽  
Arpita Bharadwaj ◽  
Richard Posert ◽  
Craig Yoshioka ◽  
Isabelle Baconguis
Keyword(s):  
Electron Microscopy ◽  
Ion Channel ◽  
Sodium Channel ◽  
Conformational Changes ◽  
Single Particle ◽  
Transmembrane Domain ◽  
Epithelial Sodium Channel ◽  
Cryo Electron Microscopy ◽  
Inhibitory Domain ◽  
Epithelial Sodium Channel Enac

The epithelial sodium channel (ENaC), a member of the ENaC/DEG superfamily, regulates Na+ and water homeostasis. ENaCs assemble as heterotrimeric channels that harbor protease-sensitive domains critical for gating the channel. Here, we present the structure of human ENaC in the uncleaved state determined by single-particle cryo-electron microscopy. The ion channel is composed of a large extracellular domain and a narrow transmembrane domain. The structure reveals that ENaC assembles with a 1:1:1 stoichiometry of α:β:γ subunits arranged in a counter-clockwise manner. The shape of each subunit is reminiscent of a hand with key gating domains of a ‘finger’ and a ‘thumb.’ Wedged between these domains is the elusive protease-sensitive inhibitory domain poised to regulate conformational changes of the ‘finger’ and ‘thumb’; thus, the structure provides the first view of the architecture of inhibition of ENaC.

scholarly journals Cryo-EM Reveals the Structural Basis of Microtubule Depolymerization by Kinesin-13s

2017 ◽  
Author(s):  
Matthieu P. M. H. Benoit ◽  
Ana B. Asenjo ◽  
Hernando Sosa
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Distinct Group ◽  
Atomic Resolution ◽  
Structural Basis ◽  
Structural Elements ◽  
Microtubule Depolymerization ◽  
Cryo Electron Microscopy ◽  
Kinesin Superfamily ◽  
Motile Activity

SummaryKinesin-13s constitute a distinct group within the kinesin superfamily of motor proteins that promotes microtubule depolymerization and lacks motile activity. The molecular mechanism by which the kinesins depolymerize microtubules and are adapted to perform a seemingly very different activity from other kinesins is still unclear. To address this issue we obtained near atomic resolution cryo-electron microscopy (cryo-EM) structures of Drosophila melanogaster kinesin-13 KLP10A constructs bound to curved or straight tubulin in different nucleotide states. The structures show how nucleotide induced conformational changes near the catalytic site are coupled with kinesin-13-specific structural elements to induce tubulin curvature leading to microtubule depolymerization. The data highlight a modular structure that allows similar kinesin core motor-domains to be used for different functions, such as motility or microtubule depolymerization.

scholarly journals Molecular basis for the ATPase-powered substrate translocation by the Lon AAA+ protease

2020 ◽  
Author(s):  
Kaiming Zhang ◽  
Shanshan Li ◽  
Kan-Yen Hsieh ◽  
Shih-Chieh Su ◽  
Grigore D. Pintilie ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Molecular Basis ◽  
Structural Basis ◽  
Cryo Electron Microscopy ◽  
Adenosine Triphosphatases ◽  
Translocation Mechanism ◽  
Atp Binding And Hydrolysis ◽  
Proteolytic Chamber ◽  
Specific Nucleotide

AbstractThe Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of the substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we have determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) at 3.6 Å resolution in a substrate-engaged state. Substrate interactions are mediated by the dual pore-loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states; a closed AAA+ ring is nevertheless maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. The structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding.

Frontiers in Cryo Electron Microscopy of Complex Macromolecular Assemblies

2019 ◽  
Vol 21 (1) ◽  
pp. 395-415 ◽  
Author(s):  
Jana Ognjenović ◽  
Reinhard Grisshammer ◽  
Sriram Subramaniam
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Specimen Preparation ◽  
Macromolecular Assemblies ◽  
Cryo Electron Microscopy ◽  
G Protein Coupled ◽  
Improved Methods

In recent years, cryo electron microscopy (cryo-EM) technology has been transformed with the development of better instrumentation, direct electron detectors, improved methods for specimen preparation, and improved software for data analysis. Analyses using single-particle cryo-EM methods have enabled determination of structures of proteins with sizes smaller than 100 kDa and resolutions of ∼2 Å in some cases. The use of electron tomography combined with subvolume averaging is beginning to allow the visualization of macromolecular complexes in their native environment in unprecedented detail. As a result of these advances, solutions to many intractable challenges in structural and cell biology, such as analysis of highly dynamic soluble and membrane-embedded protein complexes or partially ordered protein aggregates, are now within reach. Recent reports of structural studies of G protein–coupled receptors, spliceosomes, and fibrillar specimens illustrate the progress that has been made using cryo-EM methods, and are the main focus of this review.

scholarly journals Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates.

1995 ◽  
Vol 129 (5) ◽  
pp. 1311-1328 ◽  
Author(s):  
D Chrétien ◽  
S D Fuller ◽  
E Karsenti
Keyword(s):  
Electron Microscopy ◽  
Growth Rate ◽  
Conformational Changes ◽  
Two Dimensional ◽  
Closure Rate ◽  
Cryo Electron Microscopy ◽  
Stochastic Fluctuations ◽  
Microtubule Growth ◽  
The Mean ◽  
Dependent Growth

Observation of microtubule growth at different rates by cryo-electron microscopy reveals that the ends range from blunt to long, gently curved sheets. The mean sheet length increases with the growth rate while the width of the distributions increases with the extent of assembly. The combination of a concentration dependent growth rate of the tubulin sheet with a variable closure rate of the microtubule cylinder, results in a model in which stochastic fluctuations in sheet length and tubulin conformation confine GTP-tubulins to microtubule ends. We propose that the variability of microtubule growth rate observed by video microscopy (Gildersleeve, R. F., A. R. Cross, K. E. Cullen, A. P. Fagen, and R. C. Williams. 1992. J. Biol. Chem. 267: 7995-8006, and this study) is due to the variation in the rate of cylinder closure. The curvature of the sheets at the end of growing microtubules and the small oligomeric structures observed at the end of disassembling microtubules, indicate that tubulin molecules undergo conformational changes both during assembly and disassembly.

scholarly journals Antibody-induced uncoating of human rhinovirus B14

2017 ◽  
Vol 114 (30) ◽  
pp. 8017-8022 ◽  
Author(s):  
Yangchao Dong ◽  
Yue Liu ◽  
Wen Jiang ◽  
Thomas J. Smith ◽  
Zhikai Xu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Virus Infection ◽  
Conformational Changes ◽  
Neutralizing Antibody ◽  
Antigen Binding ◽  
Fab Fragment ◽  
Cryo Electron Microscopy ◽  
Virus Complex ◽  
Positive Strand Rna ◽  
Icosahedral Capsid

Rhinoviruses (RVs) are the major causes of common colds in humans. They have a nonenveloped, icosahedral capsid surrounding a positive-strand RNA genome. Here we report that the antigen-binding (Fab) fragment of a neutralizing antibody (C5) can trigger genome release from RV-B14 to form emptied particles and neutralize virus infection. Using cryo-electron microscopy, structures of the C5 Fab in complex with the full and emptied particles have been determined at 2.3 Å and 3.0 Å resolution, respectively. Each of the 60 Fab molecules binds primarily to a region on viral protein 3 (VP3). Binding of the C5 Fabs to RV-B14 results in significant conformational changes around holes in the capsid through which the viral RNA might exit. These results are so far the highest resolution view of an antibody–virus complex and elucidate a mechanism whereby antibodies neutralize RVs and related viruses by inducing virus uncoating.

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