Three-dimensional Structure of the Toxin-delivery Particle Antifeeding Prophage of Serratia entomophila

Heymann, J. Bernard; Bartho, J.D.; Rybakova, Daria; Venugopal, Hari; Winkler, Dennis C.; Sen, Abhik; Hurst, Mark R. H.; Mitra, Alok K.

doi:10.1074/jbc.m113.456145

Cited by 59 publications

(74 citation statements)

References 39 publications

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“…This process resulted in an intermediate $10-Å resolution reconstruction ( Figure S1A) that displayed clearly the helical nature of the 3D structure. We used this reconstruction to determine the helical symmetry using a real-space autocorrelation method (Heymann et al, 2013), which yielded an initial estimate for the axial rise of 42.7 Å and azimuthal rotation angle of 7.9 ( Figure S1B). The helical parameters were then refined using the iterative helical real-space reconstruction (IHRSR) approach (Egelman, 2000), which applies search and enforcement of helical symmetry, varying the values for axial rise and azimuthal rotation around the initial estimates.…”

Section: Resultsmentioning

confidence: 99%

“…C6 symmetry was imposed and the orientation search was restricted to ±10 for the out-of-plane tilt. The resulting reconstruction showed a clear helical nature, which was quantified using real-space autocorrelation methods described earlier (Heymann et al, 2013), giving initial symmetry parameters for axial rise and azimuthal rotation angle.…”

Section: Cryo-em Of Nanotubes and Image Processingmentioning

confidence: 99%

“…First, a reconstruction without helical symmetry imposition was performed in order to calculate ab initio estimates of the helical symmetry parameters as described (Heymann et al, 2013). Second, these helix parameters were refined using IHRSR and additional tools for helical symmetry determination and imposition (Egelman, 2000).…”

Section: Cryo-em Of Nanotubes and Image Processingmentioning

confidence: 99%

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Cryo-Electron Microscopy Structure of Human Peroxiredoxin-3 Filament Reveals the Assembly of a Putative Chaperone

et al. 2015

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Peroxiredoxins (Prxs) are a ubiquitous class of thiol-dependent peroxidases that play an important role in the protection and response of cells to oxidative stress. The catalytic unit of typical 2-Cys Prxs are homodimers, which can self-associate to form complex assemblies that are hypothesized to have signaling and chaperone activity. Mitochondrial Prx3 forms dodecameric toroids, which can further stack to form filaments, the so-called high-molecular-weight (HMW) form that has putative holdase activity. We used single-particle analysis and helical processing of electron cryomicroscopy images of human Prx3 filaments induced by low pH to generate a ∼7-Å resolution 3D structure of the HMW form, the first such structure for a 2-Cys Prx. The pseudo-atomic model reveals interactions that promote the stacking of the toroids and shows that unlike previously reported data, the structure can accommodate a partially folded C terminus. The HMW filament lumen displays hydrophobic patches, which we hypothesize bestow holdase activity.

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Section: Resultsmentioning

confidence: 99%

Section: Cryo-em Of Nanotubes and Image Processingmentioning

confidence: 99%

Section: Cryo-em Of Nanotubes and Image Processingmentioning

confidence: 99%

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Cryo-Electron Microscopy Structure of Human Peroxiredoxin-3 Filament Reveals the Assembly of a Putative Chaperone

et al. 2015

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“…8,309 baseplates and distal ends were identified after 2D classification, and 2,660 baseplates and 3,710 distal ends were further processed by RELION1.4 (Scheres, 2012) to obtain 3D density (Appendix Table S1). Based on available structural data of other contractile tail‐like structures (Schwarzer et al , 2012; Heymann et al , 2013; Shikuma et al , 2014; Ge et al , 2015; Nováček et al , 2016; Taylor et al , 2016), we applied a sixfold symmetry (C6) during refinement of the baseplate and the distal end. The final resolution was estimated to be on average 8.7 Å (local resolution distribution from 7.2 to 14.2 Å) for the baseplate and 7.5 Å (from 6.7 to 13.7 Å) for the distal end (Fig EV1A).…”

Section: Resultsmentioning

confidence: 99%

“…Bacteria use phage‐related contractile nanomachines to kill competition or manipulate the surrounding environment (Nakayama et al , 2000; Heymann et al , 2013; Shikuma et al , 2014; Ge et al , 2015). Evolutionarily related Type VI secretion system (T6SS) is used by bacteria to deliver proteins into both bacterial and eukaryotic cells (Mougous et al , 2006; Pukatzki et al , 2006; Hood et al , 2010; MacIntyre et al , 2010; Durand et al , 2014; Ho et al , 2014; Alcoforado Diniz et al , 2015; Hachani et al , 2016).…”

Section: Introductionmentioning

confidence: 99%

Cryo‐ EM reconstruction of Type VI secretion system baseplate and sheath distal end

et al. 2017

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The bacterial Type VI secretion system (T6SS) assembles from three major parts: a membrane complex that spans inner and outer membranes, a baseplate, and a sheath–tube polymer. The baseplate assembles around a tip complex with associated effectors and connects to the membrane complex by TssK. The baseplate assembly initiates sheath–tube polymerization, which in some organisms requires TssA. Here, we analyzed both ends of isolated non‐contractile Vibrio cholerae sheaths by cryo‐electron microscopy. Our analysis suggests that the baseplate, solved to an average 8.0 Å resolution, is composed of six subunits of TssE/F2/G and the baseplate periphery is decorated by six TssK trimers. The VgrG/PAAR tip complex in the center of the baseplate is surrounded by a cavity, which may accommodate up to ~450 kDa of effector proteins. The distal end of the sheath, resolved to an average 7.5 Å resolution, shows sixfold symmetry; however, its protein composition is unclear. Our structures provide an important step toward an atomic model of the complete T6SS assembly.

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Guidelines for using Bsoft for high resolution reconstruction and validation of biomolecular structures from electron micrographs

2017

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Cryo-electron microscopy (cryoEM) is becoming popular as a tool to solve biomolecular structures with the recent availability of direct electron detectors allowing automated acquisition of high resolution data. The Bsoft software package, developed over 20 years for analyzing electron micrographs, offers a full workflow for validated single particle analysis with extensive functionality, enabling customization for specific cases. With the increasing use of cryoEM and its automation, proper validation of the results is a bigger concern. The three major validation approaches, independent data sets, resolution-limited processing, and coherence testing, can be incorporated into any Bsoft workflow. Here, the main workflow is divided into four phases: (i) micrograph preprocessing, (ii) particle picking, (iii) particle alignment and reconstruction, and (iv) interpretation. Each of these phases represents a conceptual unit that can be automated, followed by a check point to assess the results. The aim in the first three phases is to reconstruct one or more validated maps at the best resolution possible. Map interpretation then involves identification of components, segmentation, quantification, and modeling. The algorithms in Bsoft are well established, with future plans focused on ease of use, automation and institutionalizing validation.

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Three-dimensional Structure of the Toxin-delivery Particle Antifeeding Prophage of Serratia entomophila

Cited by 59 publications

References 39 publications

Cryo-Electron Microscopy Structure of Human Peroxiredoxin-3 Filament Reveals the Assembly of a Putative Chaperone

Cryo-Electron Microscopy Structure of Human Peroxiredoxin-3 Filament Reveals the Assembly of a Putative Chaperone

Cryo‐ EM reconstruction of Type VI secretion system baseplate and sheath distal end

Guidelines for using Bsoft for high resolution reconstruction and validation of biomolecular structures from electron micrographs

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