scholarly journals Distinct architecture and composition of mouse axonemal radial spoke head revealed by cryo-EM

2021 ◽  
Vol 118 (4) ◽  
pp. e2021180118
Author(s):  
Wei Zheng ◽  
Fan Li ◽  
Zhanyu Ding ◽  
Hao Liu ◽  
Lei Zhu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Brake Pad ◽  
Core Complex ◽  
Central Pair ◽  
Radial Spoke ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Compact Body ◽  
Β Sheets ◽  
Cilia And Flagella

The radial spoke (RS) heads of motile cilia and flagella contact projections of the central pair (CP) apparatus to coordinate motility, but the morphology is distinct for protozoa and metazoa. Here we show the murine RS head is compositionally distinct from that ofChlamydomonas. Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a and Rsph10b, whose orthologs exist in the protozoan RS head. We resolve its cryo-electron microscopy (cryo-EM) structure at 3.2-Å resolution. Our atomic model further reveals a twofold symmetric brake pad-shaped structure, in which Rsph4a and Rsph9 form a compact body extended laterally with two long arms of twisted Rsph1 β-sheets and potentially connected dorsally via Rsph3b to the RS stalk. Furthermore, our modeling suggests that the core complex contacts the periodic CP projections either rigidly through its tooth-shaped Rsph4a regions or elastically through both arms for optimized RS–CP interactions and mechanosignal transduction.

scholarly journals Distinct architecture and composition of mouse axonemal radial spoke head revealed by cryo-EM

2019 ◽  
Author(s):  
Wei Zheng ◽  
Fan Li ◽  
Zhanyu Ding ◽  
Hao Liu ◽  
Lei Zhu ◽  
...  
Keyword(s):  
Interaction Network ◽  
Core Complex ◽  
Central Pair ◽  
Radial Spoke ◽  
Ciliary Motility ◽  
Axonemal Dynein ◽  
Compact Body ◽  
Dynein Arms

AbstractThe radial spoke (RS) transmits mechanochemical signals from the central pair apparatus (CP) to axonemal dynein arms to coordinate ciliary motility. The RS head, directly contacting with CP, differs dramatically in morphology between protozoan and mammal. Here we show the murine RS head is compositionally distinct from the Chlamydomonas one. Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a whose orthologue exists in the Chlamydomonas RS head. We present the unprecedented cryo-EM structure of RS head core complex at 4.5-Å resolution and identified the subunit location and their interaction network. In this complex, Rsph3b, Rsph4a, and Rsph9 forms a compact body with Rsph4a serving possibly as an assembly scaffold and Rsph3b in a location that might link the head with stalk. Interestingly, two Rsph1 subunits constitute the two stretching-arms possibly for optimized RS-CP interaction. We also propose a sawtooth model for the RS-CP interaction. Our study suggests that the RS head experiences profound remodeling to probably comply with both structural and functional alterations of the axoneme during evolution.

scholarly journals The topology of chromatin-binding domains in the NuRD deacetylase complex

2020 ◽  
Vol 48 (22) ◽  
pp. 12972-12982
Author(s):  
Christopher J Millard ◽  
Louise Fairall ◽  
Timothy J Ragan ◽  
Christos G Savva ◽  
John W R Schwabe
Keyword(s):  
Electron Microscopy ◽  
Associated Factors ◽  
Chromatin Binding ◽  
Potential Mechanism ◽  
Nurd Complex ◽  
Core Complex ◽  
The Core ◽  
Binding Domains ◽  
Cryo Electron Microscopy ◽  
Nuclear Processes

Abstract Class I histone deacetylase complexes play essential roles in many nuclear processes. Whilst they contain a common catalytic subunit, they have diverse modes of action determined by associated factors in the distinct complexes. The deacetylase module from the NuRD complex contains three protein domains that control the recruitment of chromatin to the deacetylase enzyme, HDAC1/2. Using biochemical approaches and cryo-electron microscopy, we have determined how three chromatin-binding domains (MTA1-BAH, MBD2/3 and RBBP4/7) are assembled in relation to the core complex so as to facilitate interaction of the complex with the genome. We observe a striking arrangement of the BAH domains suggesting a potential mechanism for binding to di-nucleosomes. We also find that the WD40 domains from RBBP4 are linked to the core with surprising flexibility that is likely important for chromatin engagement. A single MBD2 protein binds asymmetrically to the dimerisation interface of the complex. This symmetry mismatch explains the stoichiometry of the complex. Finally, our structures suggest how the holo-NuRD might assemble on a di-nucleosome substrate.

scholarly journals The structure of an injectisome export gate demonstrates conservation of architecture in the core export gate between flagellar and virulence type three secretion systems

2019 ◽  
Author(s):  
Steven Johnson ◽  
Lucas Kuhlen ◽  
Justin C. Deme ◽  
Patrizia Abrusci ◽  
Susan M. Lea
Keyword(s):  
Electron Microscopy ◽  
Membrane Proteins ◽  
Complex Analysis ◽  
Secretion Systems ◽  
Core Complex ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Equivalent Complex ◽  
Type Three Secretion ◽  
Resolution Structure

AbstractExport of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported though a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assemble into an extra-membrane helical assembly that likely seeds correct assembly of the rod above. Here we present the structure of an equivalent complex from the more fragile Shigella virulence system at 3.5 Å by cryo-electron microscopy. This higher resolution structure reveals further detail and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also reveals details of how the SctS/FliQ subunits sequentially assemble in the complex.

Study of eukaryotic flagellar components by cryo-electron microscopy

1986 ◽  
Vol 44 ◽  
pp. 98-101
Author(s):  
John M. Murray ◽  
Rob Ward
Keyword(s):  
Electron Microscopy ◽  
Primary Motion ◽  
Cryo Electron Microscopy ◽  
Cross Links ◽  
Cilia And Flagella

The eukaryotic flagellum is constructed from 11 parallel tubular elements arranged as 9 peripheral fibers (doublet microtubules) and 2 central fibers (singlet microtubules). The primary motion generating component has been found to be arranged as axially periodic “arms” bridging the adjacent doublets. The dynein, comprising the arms, has been isolated and characterized from several different cilia and flagella. Various radial and azimuthal cross-links stabilize the axially aligned microtubules, and probably play some role in controlling the form of the flagella beat cycle.

scholarly journals The Structure of an Injectisome Export Gate Demonstrates Conservation of Architecture in the Core Export Gate between Flagellar and Virulence Type III Secretion Systems

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Steven Johnson ◽  
Lucas Kuhlen ◽  
Justin C. Deme ◽  
Patrizia Abrusci ◽  
Susan M. Lea
Keyword(s):  
Type Iii Secretion ◽  
Complex Analysis ◽  
Secretion Systems ◽  
Core Complex ◽  
Type Iii ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Type Iii Secretion Systems ◽  
Equivalent Complex ◽  
Resolution Structure

ABSTRACT Export of proteins through type III secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported through a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assembles into an extramembrane helical assembly that likely seeds correct assembly of the rod. Here we present the structure of an equivalent complex from the Shigella virulence system at 3.5 Å by cryo-electron microscopy. This higher-resolution structure yields a more precise description of the structure and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also suggests how the SctS/FliQ subunits sequentially assemble in the complex. IMPORTANCE Although predicted on the basis of sequence conservation, the work presented here formally demonstrates that all classes of type III secretion systems, flagellar or virulence, share the same architecture at the level of the core structures. This absolute conservation of the unusual extramembrane structure of the core export gate complex now allows work to move to focusing on both mechanistic studies of type III but also on fundamental studies of how such a complex is assembled.

scholarly journals Near-atomic cryo-EM structure of yeast kinesin-5-microtubule complex reveals a distinct binding footprint

2018 ◽  
Author(s):  
Ottilie von Loeffelholz ◽  
Alejandro Peña ◽  
Douglas Robert Drummond ◽  
Robert Cross ◽  
Carolyn Ann Moores
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
Fission Yeast ◽  
Atomic Structure ◽  
Binding Pocket ◽  
Drug Binding ◽  
Motor Domain ◽  
List Type ◽  
The Core ◽  
Cryo Electron Microscopy

SummaryKinesin-5s are essential members of the superfamily of microtubule-dependent motors that undertake conserved roles in cell division. We investigated coevolution of the motor-microtubule interface using cryo-electron microscopy to determine the near-atomic structure of the motor domain of Cut7, the fission yeast kinesin-5, bound to fission yeast microtubules. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation, with a closed nucleotide binding pocket and docked neck linker that supports cover neck bundle formation. Compared to mammalian tubulin microtubules, Cut7’s footprint on S. pombe microtubule surface is subtly different because of their different architecture. However, the core motor-microtubule interaction that stimulates motor ATPase is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. The S. pombe microtubules were bound by the drug epothilone, which is visible in the taxane binding pocket. Stabilization of S. pombe microtubules is mediated by drug binding at this conserved site despite their noncanonical architecture and mechanochemistry.HighlightsS. pombe Cut7 has a distinct binding footprint on S. pombe microtubulesThe core interface driving microtubule activation of motor ATPase is conservedThe neck linker is docked in AMPPNP-bound Cut7 and the cover neck bundle is formedEpothilone binds at the taxane binding site to stabilize S. pombe microtubuleseTOC textTo investigate coevolution of the motor-microtubule interface, we used cryo-electron microscopy to determine the near-atomic structure of the motor domain of Cut7, the fission yeast kinesin-5, bound to microtubules polymerized from natively purified fission yeast tubulin and stabilised by the drug epothilone.

scholarly journals Cryo-Electron Microscopy Structure of Adenovirus Type 2 Temperature-Sensitive Mutant 1 Reveals Insight into the Cell Entry Defect

2009 ◽  
Vol 83 (15) ◽  
pp. 7375-7383 ◽  
Author(s):  
Mariena Silvestry ◽  
Steffen Lindert ◽  
Jason G. Smith ◽  
Oana Maier ◽  
Christopher M. Wiethoff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Adenovirus Type ◽  
Cell Entry ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Adenovirus Type 2

ABSTRACT The structure of the adenovirus type 2 temperature-sensitive mutant 1 (Ad2ts1) was determined to a resolution of 10 Å by cryo-electron microscopy single-particle reconstruction. Ad2ts1 was prepared at a nonpermissive temperature and contains the precursor forms of the capsid proteins IIIa, VI, and VIII; the core proteins VII, X (mu), and terminal protein (TP); and the L1-52K protein. Cell entry studies have shown that although Ad2ts1 can bind the coxsackievirus and Ad receptor and undergo internalization via αv integrins, this mutant does not escape from the early endosome and is targeted for degradation. Comparison of the Ad2ts1 structure to that of mature Ad indicates that Ad2ts1 has a different core architecture. The Ad2ts1 core is closely associated with the icosahedral capsid, a connection which may be mediated by preproteins IIIa and VI. Density within hexon cavities is assigned to preprotein VI, and membrane disruption assays show that hexon shields the lytic activity of both the mature and precursor forms of protein VI. The internal surface of the penton base in Ad2ts1 appears to be anchored to the core by interactions with preprotein IIIa. Our structural analyses suggest that these connections to the core inhibit the release of the vertex proteins and lead to the cell entry defect of Ad2ts1.

scholarly journals Shulin packages axonemal outer dynein arms for ciliary targeting

Science ◽  
2021 ◽  
Vol 371 (6532) ◽  
pp. 910-916
Author(s):  
Girish R. Mali ◽  
Ferdos Abid Ali ◽  
Clinton K. Lau ◽  
Farida Begum ◽  
Jérôme Boulanger ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Motor Activity ◽  
Compact Form ◽  
Unknown Mechanism ◽  
Closed Conformation ◽  
Cryo Electron Microscopy ◽  
Two Factors ◽  
Dynein Arms ◽  
Cilia And Flagella

The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During ciliogenesis, these ~1.8-megadalton complexes are assembled in the cytoplasm and targeted to cilia by an unknown mechanism. Here, we used the ciliateTetrahymenato identify two factors (Q22YU3 and Q22MS1) that bind ODAs in the cytoplasm and are required for ODA delivery to cilia. Q22YU3, which we named Shulin, locked the ODA motor domains into a closed conformation and inhibited motor activity. Cryo–electron microscopy revealed how Shulin stabilized this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.

scholarly journals Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope

2021 ◽  
Author(s):  
Mar Pérez-Ruiz ◽  
Mar Pulido-Cid ◽  
Juan Román Luque-Ortega ◽  
José María Valpuesta ◽  
Ana Cuervo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dna Translocation ◽  
E Coli ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Core Proteins ◽  
Dna Release ◽  
Bacterial Cytoplasm ◽  
Bacterial Peptidoglycan ◽  
T7 Bacteriophage

ABSTRACTIn most bacteriophages, the genome transport across bacterial envelopes is carried out by the tail machinery. In Podoviridae viruses, where the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins are translocated and assembled into a tube within the periplasm. T7 bacteriophage, a member from the Podoviridae family, infects E. coli gram-negative bacteria. Despite extensive studies, the precise mechanism by which this virus translocates its genome remains unknown. Using cryo-electron microscopy, we have resolved the structure two different assemblies of the T7 bacteriophage DNA translocation complex, built by core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled by interaction with gp16, resulting in a tubular structure with dimensions compatible with traversing the bacterial envelope and a channel that allows DNA passage. The structure of the gp15-gp16 complex also shows the location in gp16 of a canonical transglycosylase motif essential in the bacterial peptidoglycan layer degradation. Altogether these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which helps in the understanding at the molecular level of the mechanism involved in the T7 viral DNA release in the bacterial cytoplasm.SIGNIFICANCE STATEMENTT7 bacteriophage infects E. coli bacteria. During this process, the DNA transverses the bacterial cell wall, but the precise mechanism used by the virus remains unknown. Previous studies suggested that proteins found inside the viral capsid (core proteins) disassemble and reassemble in the bacterial periplasm to form a DNA translocation channel. In this article we solved by cryo-electron microscopy two different assemblies of the core proteins that reveal the steps followed by them to finally form a tube large enough to traverse the periplasm, as well as the location of the transglycosylase enzyme involved in peptidoglycan degradation. These findings confirm previously postulated hypothesis and make experimentally visible the mechanism of DNA transport trough the bacterial wall.

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