CTF determination and correction in electron cryotomography

Background: NAIP5 and NLRC4 induce an innate immune response to intracellular flagellin. Results: Flagellin fragments were identified that induce signaling-competent NAIP5-NLRC4 inflammasomes with 11-and 12-fold symmetry. Conclusion: Conserved flagellin terminal regions induce an inflammasome in which NAIP5 and NLRC4 appear to occupy equivalent positions. Significance: We provide fundamental insights into the formation and structure of hetero-oligomeric inflammasomes.

show abstract

“…In the case of polymerized NLRC4, the contrast transfer function was corrected by phase flipping after defocus estimation using TOMOCTF (31).…”

Section: Methodsmentioning

confidence: 99%

Formation and Structure of a NAIP5-NLRC4 Inflammasome Induced by Direct Interactions with Conserved N- and C-terminal Regions of Flagellin

Halff

Diebolder

Versteeg

et al. 2012

Journal of Biological Chemistry

144

149

View full text Add to dashboard Cite

show abstract

“…For single-particle analysis of negatively stained particles, we first reduced image frames by a factor of 2. The phase reversal due to the contrast transfer function (CTF) was corrected by taking the astigmatism into account with a modified version of CTFCORRECT from the TOMOCTF package (11). The EMAN software suite (36) was used for the following single-particle analysis.…”

Section: Methodsmentioning

confidence: 99%

Structure of the Flagellar Motor Protein Complex PomAB: Implications for the Torque-Generating Conformation

2011

View full text Add to dashboard Cite

The bacterial flagellar motor is driven by an ion flux through a channel called MotAB in Escherichia coli or Salmonella and PomAB in Vibrio alginolyticus. PomAB is composed of two transmembrane (TM) components, PomA and PomB, and converts a sodium ion flux to rotation of the flagellum. Its homolog, MotAB, utilizes protons instead of sodium ions. PomB/MotB has a peptidoglycan (PG)-binding motif in the periplasmic domain, allowing it to function as the stator by being anchored to the PG layer. To generate torque, PomAB/MotAB is thought to undergo a conformational change triggered by the ion flux and to interact directly with FliG, a component of the rotor. Here, we present the first three-dimensional structure of this torque-generating stator unit analyzed by electron microscopy. The structure of PomAB revealed two arm domains, which contain the PG-binding site, connected to a large base made of the TM and cytoplasmic domains. The arms lean downward to the membrane surface, likely representing a "plugged" conformation, which would prevent ions leaking through the channel. We propose a model for how PomAB units are placed around the flagellar basal body to function as torque generators.There are many essential biological processes coupled to ion potentials across the membrane. These include ATP synthesis by the F 1 F o -ATPase and rotation of the bacterial flagellar motor. In Escherichia coli or Salmonella, an inner membrane complex composed of two components, MotA and MotB (9,50,8,62), converts proton flux into rotation of the flagellum to power bacterial motility (2, 25, 52). The MotAB complex also functions as a stator by being anchored to the peptidoglycan (PG) layer of the cell through a binding motif in the periplasmic domain of MotB (10,23). Other channel complexes, ExbBD and TolQR, have weak sequence similarity to MotAB but utilize the proton motive force to power very different physiological functions (14,24). ExbBD is involved in active transport of iron siderophores and vitamin B 12 through the outer membrane (24), whereas TolQR is known to maintain the integrity of the outer membrane (14). Both complexes need other subunits, TonB for ExbBD or TolAB and Pal for TolQR, to bridge the periplasmic space.Some bacteria, such as alkalophilic Bacillus and Vibrio species, use an electrochemical potential of sodium ions to drive the flagellar motor (59, 39). In Vibrio species, which normally have only one polar flagellum, the four proteins PomA, PomB, MotX, and MotY are necessary to generate torque (59, 39). The maximum rotational speed of the sodium-driven motor in Vibrio alginolyticus is ϳ1,700 Hz (37), whereas the maximum speed of the proton-driven motor in E. coli is ϳ300 Hz (7, 35). PomA and PomB are closely related to MotA and MotB, respectively, whereas paralogs of MotX and MotY (40,41,44) are not found in E. coli. Although the functions of MotX and MotY are not yet clear, it has been shown that they form a ring structure, called the T ring, that protrudes into the periplasmic space ( Fig. 1), where it coul...

show abstract

“…The thickness of the final reconstructed volume contains up to one half of the viral particle. We took advantage of the contrasted images to individually detect and correct the CTF (contrast transfer function) (see Materials and methods section) (Fernandez et al, 2006), avoiding the aberrations that small defocus variations along the tilted series could introduce in the final volume. Also, denoising algorithms were applied to the reconstructed volumes, with the aim of a better discernment of the viral membranes continuity (Fernandez and Li, 2003;Cyrklaff et al, 2005;Fernandez and Li, 2005).…”

Section: Early IV Structurementioning

confidence: 99%

“…These resolutions were between 35 and 45 Å . The CTF of the images was corrected using a new approach of the TOMOCTF software package (Fernandez et al, 2006). This software computes the defocus using strips extracted from the images in the tilted series.…”

Section: Electron Tomography and Reconstructionmentioning

confidence: 99%

Membrane remodelling during vaccinia virus morphogenesis

Chichón

Rodriguez

Risco

et al. 2009

Biology of the Cell

View full text Add to dashboard Cite

Background information. VACV (vaccinia virus) is one of the most complex viruses, with a size exceeding 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and important rearrangements of its structural components.Results. We have used electron tomography of sections of VACV-infected cells to follow, in three dimensions, the remodelling of the membrane components of the virus during envelope maturation. The tomograms obtained suggest that a number of independent 'crescents' interact with each other to enclose the volume of an incomplete ellipsoid in the viral factory area, attaining the overall shape and size characteristic of the first immature form of the virus [IV (immature virus)]. The incorporation of the DNA into these forms leads to particles with a nucleoid [IVN (IV with nucleoid)] that results in local disorganization of the envelope in regions near the condensed DNA. These particles suffer the progressive disappearance of the membrane outer spikes with a change in the shape of the membrane, becoming locally curled. The transformation of the IVN into the mature virus involves an extreme rearrangement of the particle envelope, which becomes fragmented and undulated. During this process, we also observed connections between the outer membranes with internal ones, suggesting that the latter originate from internalization of the IV envelope.Conclusions. The main features observed for VACV membrane maturation during morphogenesis resemble the breakdown and reassembly of cellular endomembranes.

show abstract

CTF determination and correction in electron cryotomography

Cited by 188 publications

References 36 publications

Formation and Structure of a NAIP5-NLRC4 Inflammasome Induced by Direct Interactions with Conserved N- and C-terminal Regions of Flagellin

Formation and Structure of a NAIP5-NLRC4 Inflammasome Induced by Direct Interactions with Conserved N- and C-terminal Regions of Flagellin

Structure of the Flagellar Motor Protein Complex PomAB: Implications for the Torque-Generating Conformation

Membrane remodelling during vaccinia virus morphogenesis

Contact Info

Product

Resources

About