Parakeet: a digital twin software pipeline to assess the impact of experimental parameters on tomographic reconstructions for cryo-electron tomography

In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a ‘digital twin’ software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.     

Cryo electron tomography of vitrified fibroblasts: microtubule plus ends in situ

Mouse embryonic fibroblasts (MEFs) are cells that have highly suitable biophysical properties for cellular cryo electron tomography. MEFs can be grown directly on carbon supported by EM grids. They stretch out and grow thinner than 500 nm over major parts of the cell, attaining a minimal thickness of 50 nm at their cortex. This facilitates direct cryo-fixation by plunge-freezing and high resolution cryo electron tomography. Both by direct cryo electron microscopy projection imaging and cryo electron tomography of vitrified MEFs we visualized a variety of cellular structures like ribosomes, vesicles, mitochondria, rough endoplasmatic reticulum, actin filaments, intermediate filaments and microtubules.MEFs are primary cells that closely resemble native tissue and are highly motile. Therefore, they are attractive for studying cytoskeletal elements. Here we report on structural investigations of microtubule plus ends. We were able to visualize single frayed protofilaments at the microtubule plus end in vitrified fibroblasts using cryo electron tomography. Furthermore, it appeared that MEFs contain densities inside their microtubules, although 2.5–3.5 times less than in neuronal cells [Garvalov, B.K., Zuber, B., Bouchet-Marquis, C., Kudryashev, M., Gruska, M., Beck, M., Leis, A., Frischknecht, F., Bradke, F., Baumeister, W., Dubochet, J., and Cyrklaff, M. 2006. Luminal particles within cellular microtubules. J. Cell Biol. 174, 759–765]. Projection imaging of cellular microtubule plus ends showed that 40% was frayed, which is two times more than expected when compared to microtubule growth and shrinkage rates in MEFs. This suggests that frayed ends might be stabilized in the cell cortex.     

Structure of Halothiobacillus neapolitanus carboxysomes by cryo-electron tomography

Carboxysomes are polyhedral bodies consisting of a proteinaceous shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). They are found in the cytoplasm of all cyanobacteria and some chemoautotrophic bacteria. Previous studies of Halothiobacillus neapolitanus and Nitrobacter agilis carboxysomes suggest that the structures are either icosahedral or dodecahedral. To determine the protein shell structure more definitively, purified H. neapolitanus carboxysomes were re-examined by cryo-electron tomography and scanning transmission electron microscopy (STEM). Due to the limited tilt angles in the electron microscope, the tomographic reconstructions are distorted. Corrections were made in the 3D orientation searching and averaging of the computationally extracted carboxysomes to minimize the missing data effects. It was found that H. neapolitanus carboxysomes vary widely in size and mass as shown by cryo-electron tomography and STEM mass measurements, respectively. We have aligned and averaged carboxysomes in several size classes from the 3D tomographic reconstruction by methods that are not model-biased. The averages reveal icosahedral symmetry of the shell, but not of the density inside it, for all the size classes.     

Implementation of a flash-photolysis system for time-resolved cryo-electron microscopy

We describe here the implementation of a flash-photolysis system for time-resolved cryo-electron microscopy. A previously designed computer-controlled cryo-plunging apparatus [White, H.D., Thirumurugan, K., Walker, M.L., Trinick, J., 2003. A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray. J. Struct. Biol. 144, 246–252] was used as a hardware platform, onto which a xenon flash lamp and liquid light pipe were mounted. The irradiation initiates a reaction through cleavage of the photolabile blocking group from a biologically active compound. The timespan between flashing and freezing in cryogen is on the order of milliseconds, and defines the fastest observable reaction. Blotting of excess fluid, which takes on the order of 1 s, is done before irradiation and thus does not represent a rate-limiting step. A specimen-heating problem, identified by measurements with a thermocouple, was alleviated with the use of thick, aluminum-coated grids.     

Bilateral edge filter: photometrically weighted, discontinuity based edge detection

Edge-detection algorithms have the potential to play an increasingly important role both in single particle analysis (for the detection of randomly oriented particles), and in tomography (for the segmentation of 3D volumes). However, the majority of traditional linear filters are significantly affected by noise as well as artefacts, and offer limited selectivity. The Bilateral edge filter presented here is an adaptation of the Bilateral filter [Jiang, W., Baker, M.L., Wu, Q., Bajaj, C., Chiu, W., 2003. Applications of a bilateral denoising filter in biological electron microscopy. J. Struct. Biol. 144, 114-122] designed for enhanced edge detection. It uses photometric weighting to identify significant discontinuities (representing edges), minimizing artefacts and noise. Compared with common edge-detectors (LoG, Marr-Hildreth) the Bilateral edge filter yielded significantly better results. Indeed data was of a similar quality to that of the Canny edge-detector, which is considered as a leading standard in edge detection [Basu, M., 2002. Gaussian-based edge-detection methods-a survey. IEEE Trans. Syst. Man Cybern. C Appl. Rev. 32, 252-260]. Compared to the Canny edge-detector the Bilateral edge-detector has the advantages that it only requires the adjustment of a single parameter, is theoretically faster for reasonably sized images, and can be used in selective contrast enhancement of images. The simplicity and speed of the filter for single particle and tomographic analysis are discussed.     

The fourfold way of the genetic code

We describe a compact representation of the genetic code that factorizes the table in quartets. It represents a “least grammar” for the genetic language. It is justified by the Klein-4 group structure of RNA bases and codon doublets. The matrix of the outer product between the column-vector of bases and the corresponding row-vector V^T = (C G U A), considered as signal vectors, has a block structure consisting of the four cosets of the K × K group of base transformations acting on doublet AA. This matrix, translated into weak/strong (W/S) and purine/pyrimidine (R/Y) nucleotide classes, leads to a code table with mixed and unmixed families in separate regions. A basic difference between them is the non-commuting (R/Y) doublets: AC/CA, GU/UG. We describe the degeneracy in the canonical code and the systematic changes in deviant codes in terms of the divisors of 24, employing modulo multiplication groups. We illustrate binary sub-codes characterizing mutations in the quartets. We introduce a decision-tree to predict the mode of tRNA recognition corresponding to each codon, and compare our result with related findings by Jestin and Soulé [Jestin, J.-L., Soulé, C., 2007. Symmetries by base substitutions in the genetic code predict 2′ or 3′ aminoacylation of tRNAs. J. Theor. Biol. 247, 391–394], and the rearrangements of the table by Delarue [Delarue, M., 2007. An asymmetric underlying rule in the assignment of codons: possible clue to a quick early evolution of the genetic code via successive binary choices. RNA 13, 161–169] and Rodin and Rodin [Rodin, S.N., Rodin, A.S., 2008. On the origin of the genetic code: signatures of its primordial complementarity in tRNAs and aminoacyl-tRNA synthetases. Heredity 100, 341–355], respectively.     

BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs

An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W., Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525–532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, V.I., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66, 331–341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1' in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high β sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae.     

Exploring tree-building methods and distinct molecular data to recover a known asymmetric phage phylogeny

An experimental phylogeny was constructed using bacteriophage T7 and a propagation protocol, in the presence of the mutagen N-methyl-N′-nitro-N′-nitrosoguanidine, based on Hillis et al. [Hillis, D.M., Bull, J.J., White, M.E., Badgett, M.R., Molineux, I.J., 1992. Experimental phylogenetics, generation of a known phylogeny. Science 255, 589–592]. The topology presented in this study has a considerable variation in branch lengths and is less symmetric than the one presented by Hillis et al. [Hillis, D.M., Bull, J.J., White, M.E., Badgett, M.R., Molineux, I.J., 1992. Experimental phylogenetics, generation of a known phylogeny. Science 255, 589–592]. These features are known to present additional difficulties to phylogenetic inference methods. The performance of several phylogenetic methods (conventional and less conventional) was tested using restriction site and nucleotide data. Only methods that encompassed a molecular clock or those based on sequence signatures recovered the true phylogeny. Nevertheless a likelihood ratio test rejected the hypothesis of the existence of a molecular clock when the whole sequence data set was considered. This fact or the particular substitution pattern (mainly G → A and C → T) may be related to the unexpected performance of distance methods based on sequence signatures. To test if the results could have been predicted by simulation studies we estimated the evolution parameters from the real phylogeny and used them to simulate evolution along the same tree (parametric bootstrap). We found that simulation could predict most but not all of the problems encountered by phylogenetic inference methods in the real phylogeny. Short interior branches may be more prone to error than predicted by theoretical studies.     

Proteolytic Cleavage Driven by Glycosylation

Proteolytic processing of human host cell factor 1 (HCF-1) to its mature form was recently shown, unexpectedly, to occur in a UDP-GlcNAc-dependent fashion within the transferase active site of O-GlcNAc-transferase (OGT) (Lazarus, M. B., Jiang, J., Kapuria, V., Bhuiyan, T., Janetzko, J., Zandberg, W. F., Vocadlo, D. J., Herr, W., and Walker, S. (2013) Science 342, 1235–1239). An interesting mechanism involving formation and then intramolecular rearrangement of a covalent glycosyl ester adduct of the HCF-1 polypeptide was proposed to account for this unprecedented proteolytic activity. However, the key intermediate remained hypothetical. Here, using a model enzyme system for which the formation of a glycosyl ester within the enzyme active site has been shown unequivocally, we show that ester formation can indeed lead to proteolysis of the adjacent peptide bond, thereby providing substantive support for the mechanism of HCF-1 processing proposed.     

Morphological and biochemical characterization of light and heavy myelin isolated from developing rat brain

Myelin from developing rat brains was separated on a discontinuous sucrose gradient into subfractions of two different densities, i.e. light and heavy myelin. Electron photomicrographs showed that heavy myelin consisted primarily of large compacted multilamellar structures with a distinct intraperiod line characteristic of myelin in situ. Light myelin, on the other hand, was composed of small vesicles having a unilamellar structure. Similar to whole myelin, both membrane subfractions were highly enriched in 2′,3′-cyclic nucleotide-3′-phosphohydrolase. The specific activity of the enzyme, however, showed no developmental trend. Both subfractions contained all of the four major proteins characteristic of the whole myelin membrane. There were, however, quantitative differences in the relative distribution of these proteins between light and heavy myelin. Basic protein accounted for 55 % and proteolipid protein for 46 % of the total myelin proteins of light and heavy myelin, respectively. DM-20 (Agrawal, H. C., Burton, R. M., Fishman, M. A., Mitchell, R. F. and Prensky, A. L. (1972) J. Neurochem. 19, 2083–2089) exhibited a developmental “switch” between light and heavy myelin. Light myelin appeared to contain more DM-20 in 15- to 20-day-old rat brain, whereas the concentration of this protein was higher in heavy myelin at subsequent ages studied.     

Absence of pyridoxine- (pyridoxamine-) 5'-phosphate oxidase in Morris hepatoma 7777

Morris hepatoma 7777 previously has been shown to have no detectable pyridoxine- (pyridoxamine-) 5'-phosphate oxidase activity [Thanassi, J. W., Nutter, L. M., Meisler, N. T., Commers, P., & Chiu, J.-F. (1981) J. Biol. Chem. 256, 3370-3375]. In order to determine if this enzyme was missing in the hepatoma, we purified rat liver oxidase and raised antibodies to it in rabbits. Final purification of rat liver oxidase for use as an antigen was accomplished by affinity chromatography and gel electrophoresis. The rat liver enzyme is similar to rabbit liver oxidase [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436-3442] having two noncovalently linked subunits with molecular weights in the range of 25 000-28 000. Evidence indicating that inactive enzyme was simultaneously purified with native enzyme was obtained. The IgG fraction was purified from the serum of a rabbit that had been immunized with rat liver oxidase. This was used in the development of ELISA and immunoblot analyses for the presence of antigenically active pyridoxine- (pyridoxamine-) 5'-phosphate oxidase in cytosolic preparations from normal rat liver and Morris hepatoma 7777. The results indicated that there was no immunologically detectable oxidase protein in the tumor. An alternate pathway of pyridoxal 5'-phosphate synthesis, involving oxidation of pyridoxine to pyridoxal followed by phosphorylation, was ruled out. The implications of these findings with respect to acquisition of nutrients by tumors are discussed.     

Dynamic regulation of the inducible nitric-oxide synthase by NO: comparison with the endothelial isoform

We studied by ultrafast time-resolved absorption spectroscopy the geminate recombination of NO to the oxygenase domain of the inducible NO synthase, iNOSoxy, and to mutated proteins at position Trp-457. This tryptophan interacts with the tetrahydrobiopterin cofactor BH4, and W457A/F mutations largely reduced the catalytic formation of NO. BH4 decreases the rate of NO rebinding to the ferric iNOSoxy compared with that measured in its absence. The pterin has a larger effect on W457A/F than on the WT protein by increasing NO release from the protein. Therefore, BH4 raises the energy barrier for NO recombination to the mutated proteins in contrast with our observations on eNOS (Slama-Schwok, A., Négrerie, M., Berka, V., Lambry, J.-C., Tsai, A.-L., Vos, M., and Martin, J.-L. (2002) J. Biol. Chem. 277, 7581-7586). Thus, we show a differential effect of BH4 on NO release from eNOS and iNOS. Compared with the position of this residue in the BH4-repleted enzyme, simulations of the NO dissociation dynamics point out at a swing of Trp-457 toward the missing pterin in the absence of BH4. NO geminate-rebinding data show a more efficient NO release from eNOS than from iNOS once NO is formed. Consistently, NO produced by iNOS is regulated by its ferric nitrosyl complex in contrast with eNOS. We show that the small enhancement of the NO geminate recombination rate in W457A/F compared with that in the WT enzyme cannot explain the decrease of NO yield because of the mutation; the major effect of the mutation thus arises from an uncoupled catalysis (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819-12825).     

Comparison of three standard anatomical reference frames for the tibia-fibula complex

Definition of anatomical reference frames is necessary both for in vitro biomechanical testing, and for in vivo human movement analyses. Different reference frames have been proposed in the literature for the lower limb, and in particular for the tibia–fibula complex. The scope of this work was to compare the three most commonly referred proposals (proposed by [Ruff, C.B., Hayes, W.C., 1983. Cross-sectional geometry at Pecos Pueblo femora and tibiae —A biomechanical investigation: I. method and general patterns of variation. American Journal of Physical Anthropology 60, pp. 359–381.], by [Cappozzo, A., Catani, F., Della Croce, U., Leardini, A., 1995. Position and orientation in space of bones during movement: anatomical frame definition and determination. Clinical Biomechanics (Bristol, Avon) 10, pp. 171–178.], and by the Standardization and Terminology Committee of the International Society of Biomechanics, [Wu, G., Siegler, S., Allard, P., Kirtley, C., Leardini, A., Rosenbaum, D., Whittle, M., D'Lima, D.D., Cristofolini, L., Witte, H., Schmid, O., Stokes, I., 2002. ISB recommendation on definitions of joint coordinate system of various joints for reporting of human joint motion—part I: ankle, hip and spine. International Society of Biomechanics. Journal of Biomechanics 35, pp. 543–548.]). These three frames were identified on six cadaveric tibia–fibula specimens based on the relevant anatomical landmarks, using a high-precision digitizer. The intra-operator (ten repetitions) and inter-operator (three operators) repeatability were investigated in terms of reference frame orientation. The three frames had similar intra-operator repeatability. The reference frame proposed by Ruff et al. had a better inter-operator repeatability (this must be put in relation with the original context of interest, i.e. in vitro measurements on dissected bones). The reference frames proposed by Ruff et al. and by ISB had a similar alignment; the frame proposed by Cappozzo et al. was considerably externally rotated and flexed with respect to the other two. Thus, the reference frame proposed by Ruff et al. is preferable when the full bone surface is accessible (typically during in vitro tests). Conversely, no advantage in terms of repeatability seems to exist between the reference frames proposed by Cappozzo et al. and ISB.     

Cryo-electron tomography and 3-D analysis of the intact flagellum in Trypanosoma brucei

Trypanosoma brucei is a uni-cellular protist that causes African sleeping sickness. These parasites have a flagellum that is attached to the cell body and is indispensible for its motility. The flagellum consists of a canonical 9+2 axoneme and a paraflagellar rod (PFR), an intricate tripartite, fibrous structure that is connected to the axoneme. In this paper we describe results from cryo-electron tomography of unperturbed flagella. This method revealed novel structures that are likely involved in attaching the flagellum to the cell. We also show the first cryo-electron tomographic images of a basal body in situ, revealing electron dense structures inside its triplet microtubules. Sub-tomogram averaging of the PFR revealed that its distal region is organized as an orthorhombic crystal.     

Electron tomography of vitreous sections from cultured mammalian cells

Cryo-electron tomography of appropriately thin, frozen-hydrated biological specimens has excellent potential for investigating the 3D macromolecular architecture of eukaryotic cells and tissues. Since cardiomyocytes are too thick to be visualised in an intact state, we grew immortalised cell line HL-1 to sub-confluency and harvested the cells by enzymatic detachment prior to hyperbaric freezing, ultramicrotomy, and tomography. We improved the efficiency of tomographic acquisition from vitreous cryosections by implementing two new features: (1) fluorescence microscopy at cryogenic temperatures to search for features of interest without expending any of the tolerable electron dose on secondary (non-imaging) tasks, and (2) the use of colloidal gold as fiducial markers. Vital fluorescent staining and subsequent cryo-fluorescence microscopy of vitreous sections were used to localise mitochondria lying in positions suitable for acquiring tilt series, taking into account section flatness, presence of contamination and proximity to grid bars. To provide a simple and robust means of aligning tomograms, we developed a universally applicable protocol for depositing colloidal gold onto vitreous sections, analogous to the method for applying quantum dots described by Masich et al. [Masich, S., Östberg, T., Norlén, L., Shupliakov, O., Daneholt, B., 2006. A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography. J. Struct. Biol. 156, 461–468]. Tomograms of thin sections (nominal thickness 65–85 nm) of cardiac mitochondria revealed the interconnectivity of cristae and junctions with the inner mitochondrial membrane. In some cases, ATP synthases could be identified without ambiguity. These findings confirm the feasibility of investigating the structural biology of mammalian cells in three dimensions and at a resolution of 6–8 nm.     

Cryo-electron tomography of Kaposi's sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures essential to capsid assembly and maturation

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a recently discovered DNA tumor virus that belongs to the γ-herpesvirus subfamily. Though numerous studies on KSHV and other herpesviruses, in general, have revealed much about their multilayered organization and capsid structure, the herpesvirus capsid assembly and maturation pathway remains poorly understood. Structural variability or irregularity of the capsid internal scaffolding core and the lack of adequate tools to study such structures have presented major hurdles to earlier investigations employing more traditional cryo-electron microscopy (cryoEM) single particle reconstruction. In this study, we used cryo-electron tomography (cryoET) to obtain 3D reconstructions of individual KSHV capsids, allowing direct visualization of the capsid internal structures and systematic comparison of the scaffolding cores for the first time. We show that B-capsids are not a structurally homogenous group; rather, they represent an ensemble of “B-capsid-like” particles whose inner scaffolding is highly variable, possibly representing different intermediates existing during the KSHV capsid assembly and maturation. This information, taken together with previous observations, has allowed us to propose a detailed pathway of herpesvirus capsid assembly and maturation.     

Books

Books reviewed in this article:
B aker . K. 1993. Identification Guide to European Non-passerines.
B arnard . C., G ilbert , F. & M c G regor
B askett , T.S., S ayre . M.W., T omlinson , R.E. & M irarchi .
B ezzel . E. 1993. Kompendium der Vögel Mitteleuropas.
B right , M. 1993. The Private Life of Birds.
C ook , M. 1992. The Birds of Moray and Nairn.
D avison . G.W.H. 1992. Birds of Mount Kinabalu. Borneo.
E rritzoe . J. 1993. The Buds of CITES and How to Identify Them.
F arner , D.S., K ing , J.R. & P arkes , K.C.
G ibbons , D.W., R eid , J.B. & C hapman . R.A. (eds). 1993. The New Atlas of Breeding Birds in Britain and Ireland.
H illman , J.C.
H uxley . E.
J ackson . C.E. 1993. Great Bird Paintings of the World.
J ohnsgard . P.A. 1993. Cormorants, Darters and Pelicans of the World.
M adge . S. & B urn , H. 1994. Crows and Jays. A Guide to the Crows, Jays and Magpies of the World.
N icolai . B. (ed.).
P ower , D.M. (ed.).
P riklonskiy . S.G. (ed.).
R alph . R. 1993. William MacGillivray.
R obinson , D. & C hapman , A.
S harp . P.J. 1993. Avian Endocrinology.
S mith , K.W., D fe , C.W., F earnside . J.D., F letcher , E.W. & S mith , R.N.
S olomon . D. & W illiams , J.
S ørensen , S., B loch . D. & L angvad . S.
Z immerman , J.L.