The V-Antigen of
            <i>Yersinia</i>
            Forms a Distinct Structure at the Tip of Injectisome Needles

Mueller, Catherine A.; Brož, Petr; Müller, Shirley A.; Ringler, Philippe; Erne-Brand, Françoise; Sorg, Isabel; Kuhn, Marina; Engel, Andréas; Cornelis, Guy R.

doi:10.1126/science.1118476

Cited by 321 publications

(375 citation statements)

References 15 publications

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“…As illustrated by the above examples, quantitative biological STEM is a welldeveloped and invaluable technology for [51] nano-analytics. As it is possible to associate mass with the shape of individual protein complexes (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Biological Scanning Transmission Electron Microscopy: Imaging and Single Molecule Mass Determination

Müller¹,

Engel²

2006

Chimia

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scanning transmission electron microscopes can both measure the mass of single protein complexes and take amazingly clear images thereof, offering a wide range of applications in structural biology. The principle of mass measurement is presented and discussed and the scope of scanning transmission electron microscopy is illustrated by selected examples. Keywords:Mass determination · scanning transmission electron microscopy · sTEM unstained proteins to be clearly visualised under low dose conditions. Each pixel of the digital image is a quantitative scattering experiment from which the mass of the irradiated volume can be calculated [9]. Thus, the mass of a single protein complex can be determined by integrating over all pixel values that lie within its boundary. The method is applicable to macromolecules of widely varying mass and form (reviewed in [10]). When combined with SDS-gel electrophoresis and, where available, sequence information, STEM mass measurements serve to define protein stoichiometry. Further, as electron energy loss spectroscopy [11][12] or energy dispersive X-ray spectroscopy [13] can be performed while imaging, the STEM also offers the possibility of determining the distribution of specific elements in protein complexes. Indeed the feasibility of mapping single atoms bound to protein molecules has recently been demonstrated [14].The high contrast delivered by the dark-field detector system [15] can also be exploited in negative or positive stain microscopy [16]. Here the STEM yields clear single-shot images that are free from phase contrast fringes. These images frequently provide information only otherwise visible after averaging several hundred projections recorded with a transmission electron microscope (TEM). They thus make it possible to document the presence of distinct protein conformations, information that would be lost by averaging techniques. Indeed, STEM images of both negatively stained [17] and unstained [18] protein complexes have been used to reconstruct their 3D structure.The few dedicated STEMs employed in biology today are invaluable. In particular, the coupling of image and mass gives them the power to answer questions not resolvable by ultracentrifugation or mass spectrometry. Here we discuss the use of the STEM both as an imaging tool and to measure mass, and examine the uncertainties to be expected in STEM mass data sets. The Importance and Versatility of STEM Mass MeasurementsWorldwide, only four laboratories routinely use dedicated STEMs to measure the mass of biological samples. Their facilities are open for external collaborations and the many papers in the literature containing STEM mass measurements document the importance of this rare technique to the scientific community. Although the precision of STEM cannot match that of the mass spectrometer, its capability to measure an enormously wide mass range, its independence from shape assumptions and the presence of an image make the STEM an invaluable tool.The methodology of mass spectrometry has develop...

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Section: Resultsmentioning

confidence: 99%

“…In a recent study carried out to investigate the structure of the needle, STEM images clearly revealed a complex at Mass [MDa] Mass [MDa] its tip ( Fig. 3a; [51]). Combined with further negative stain STEM, mutagenesis and immuno-labeling experiments showed this to be formed by the protein LcrV (also known as V-Antigen).…”

Section: Applications Of Stemmentioning

confidence: 99%

Biological Scanning Transmission Electron Microscopy: Imaging and Single Molecule Mass Determination

Müller¹,

Engel²

2006

Chimia

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“…Needle chaperones bind to needle subunits in the bacterial cytosol to prevent the physiologically unfavorable process of premature polymerization. In the absence of respective chaperones, needle proteins are rapidly degraded (8,40,41). We therefore chose to investigate potential functional implications of the FIG.…”

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confidence: 99%

Bioinformatic and Biochemical Evidence for the Identification of the Type III Secretion System Needle Protein ofChlamydia trachomatis

et al. 2008

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Chlamydia spp. express a functional type III secretion system (T3SS) necessary for pathogenesis and intracellular growth. However, certain essential components of the secretion apparatus have diverged to such a degree as to preclude their identification by standard homology searches of primary protein sequences. One example is the needle subunit protein. Electron micrographs indicate that chlamydiae possess needle filaments, and yet database searches fail to identify a SctF homologue. We used a bioinformatics approach to identify a likely needle subunit protein for Chlamydia. Experimental evidence indicates that this protein, designated CdsF, has properties consistent with it being the major needle subunit protein. CdsF is concentrated in the outer membrane of elementary bodies and is surface exposed as a component of an extracellular needle-like projection. During infection CdsF is detectible by indirect immunofluorescence in the inclusion membrane with a punctuate distribution adjacent to membrane-associated reticulate bodies. Biochemical cross-linking studies revealed that, like other SctF proteins, CdsF is able to polymerize into multisubunit complexes. Furthermore, we identified two chaperones for CdsF, termed CdsE and CdsG, which have many characteristics of the Pseudomonas spp. needle chaperones PscE and PscG, respectively. In aggregate, our data are consistent with CdsF representing at least one component of the extended Chlamydia T3SS injectisome. The identification of this secretion system component is essential for studies involving ectopic reconstitution of the Chlamydia T3SS. Moreover, we anticipate that CdsF could serve as an efficacious target for anti-Chlamydia neutralizing antibodies.

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“…The structure of MxiH, combined to the EM 3D reconstructions, allowed the building of an atomic model (Deane et al, 2006). Scanning-TEM (STEM) analyses of Y. enterocolitica needles showed that they end with a distinct tip structure consisting of a homopentamer of the protein LcrV (Mueller et al, 2005;Deane et al, 2006;Broz et al, 2007) the crystal structure of which is known (Derewenda et al, 2004). An active injectisome in contact with a target cell terminates with a translocation pore that is inserted in the plasma membrane of the target cell (Hakansson et al, 1996;Blocker et al, 1999;Neyt and Cornelis, 1999).…”

Section: The Needlementioning

confidence: 99%

The type III secretion injectisome, a complex nanomachine for intracellular ‘toxin’ delivery

Cornelis

2010

Biological Chemistry

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108

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The type III secretion injectisome is a nanomachine that delivers bacterial proteins into the cytosol of eukaryotic target cells. It consists of a cylindrical basal structure spanning the two bacterial membranes and the peptidoglycan, connected to a hollow needle, eventually followed by a filament (animal pathogens) or to a long pilus (plant pathogens). Export employs a type III pathway. During assembly, all the protein subunits of external elements are sequentially exported by the basal structure itself, implying that the export apparatus can switch its substrate specificity over time. The length of the needle is controlled by a protein that it also secreted during assembly and presumably acts as a molecular ruler.

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The V-Antigen of Yersinia Forms a Distinct Structure at the Tip of Injectisome Needles

Cited by 321 publications

References 15 publications

Biological Scanning Transmission Electron Microscopy: Imaging and Single Molecule Mass Determination

Biological Scanning Transmission Electron Microscopy: Imaging and Single Molecule Mass Determination

Bioinformatic and Biochemical Evidence for the Identification of the Type III Secretion System Needle Protein ofChlamydia trachomatis

The type III secretion injectisome, a complex nanomachine for intracellular ‘toxin’ delivery

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