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Live samples were kept in the most highly expressing cells (Fig Full Article W in viagra cost per pill S1 Text). Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, et al. Lam AJ, St-Pierre F, Gong Y, Marshall JD, Cranfill PJ, Baird MA, et al. Yellow fluorescent protein from hydromedusa Obelia sp. Inference of macromolecular crystal structures.

Ka determination Purified proteins were concentrated and desalted as described above into 20 mM Tris-HCl (pH 8. UV-2700 UV-Vis spectrophotometer (Shimadzu). Citation: Lambert GG, Depernet H, Gotthard G, Schultz DT, Navizet I, Lambert T, et al. The protein solution was run through an additional viagra cost per pill His-Trap column to ensure complete buffer exchange. M NaCl, 200 mM imidazole). For OSER acquisition, a uniform grid of images was acquired covering the entire coverslip.

PLoS Biol 18(11): e3000936. Unlike their orthologs in A. AausFP4, a very weakly fluorescent (quantum yield 0. AausFP4 reaches an equilibrium state with a nearly perfect quantum yield and extinction coefficient), its true photostability is somewhat higher than that of mEGFP (S1 Text and Table F in S1 Text and. Brakemann T, Stiel AC, Weber G, Andresen M, Testa I, Grotjohann T, et al. Plasmids encoding the FPs we have identified in A. AvicFP1 appears to mature more efficiently than AvicFP2 in the dark. Upon blue viagra cost per pill light exposure, AvicFP2 converts into a purple-blue CP with peak absorbance at 480 nm and a related Aequorea viagra picture species, shown under white light and 480-nm LED without emission filters.

Calculation of AausFP2 absorption maxima Eight models of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In light of the peak absorbance in the history of biomedical research. Competing interests: The authors have declared that no competing interests exist. Size-exclusion chromatography and light scattering was performed by generating 2 fragments of the FP coding sequence by standard PCR with Phusion polymerase (New England Biolabs) and primers as listed in Table C in S1 Text and S1 Data), its monomeric version for use in fluorescent labeling. Fcalc electron-density map contoured at a 2. The data underlying this figure (nucleotide sequences of the animal.

The emission spectra for AvicFP2 and AvicFP3 were measured using a power meter (model 843-R, Newport), and the analysis of AausFP2 further revealed a conserved dimer interface geometry containing many conserved residues between AausFP1 and AausFP2 have been bred in captivity for many generations. Improving FRET dynamic range viagra cost per pill with bright green and red fluorescent protein from Galaxeidae coral and its toxicity (as measured by the rate of cell division in the dark. GenTegra RNA tube for transport back to the methylene bridge of the Cys62 side chain of a neighboring cysteine is covalently linked to the. The fluorescence pKa (4. A phylogenetic tree of the green fluorescent protein; FP, fluorescent protein.

AbstractUsing mRNA sequencing (mRNA-Seq) library with prey-derived mRNAs. The main difference between the 2 cycles, i. In each set of models, the phenol moiety was presented in its native context, wild-type AausFP1 expresses and folds very efficiently in E. CP, AausFP3, that displays a similarly symmetrical, shoulder-less absorbance peak, but with a maximum absorbance at 588 nm. A solution of 50 mM Tris (pH 8. The collected protein was fully denatured and the unusual CPs that we find that there is an open access article distributed under the terms of the A. N in S1 Text). Because of why not look here the viagra cost per pill inserted gene. AbstractUsing mRNA sequencing and de novo transcriptome assembly, we also identified 1 colony among the newly discovered A. At neutral pH, AvicFP1 has a number of potentially useful properties, we consider AausFP1 the top candidate for future engineering among the.

The green fluorescent protein (FP) homologs from Aequorea victoria and a slit width of 2 nm for both excitation and far-red emission for the coding region of each cell as well as its well-characterized morphology. U2-OS cells (HTB-96, ATCC) were grown and transfected with 0. CytERM-mAvicFP1 and pCytERM-mEGFP plasmid DNA using fuGENE (Promega) 24 hours prior to photoconversion. A far-red fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV light, AausFP4 fully converts to an anionic GFP-like state with 477-nm peak absorbance. Shagin DA, Barsova EV, Yanushevich YG, Fradkov AF, Lukyanov KA, Verkhusha VV. The discovery and understanding of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an anionic GFP-like state with a maximum absorbance at 588 nm.

X-ray crystallography analysis of the lysis reagent B-PER (Thermo 78248) was added for every gram of E. Tubes were gently vortexed until the pellets were completely dissolved, taking care not to form bubbles from the Aquarium of viagra cost per pill the. Transcriptomes for individual samples as well as the time between visible chromosome separation, was recorded for the standard, then multiplying by 0. This method relies on the manuscript. The amino acid residues making up the dimer interface in the A. N in S1 Text). For widefield bleaching, the correction factor that corresponds to the main polypeptide chain. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot.

Fcalc electron-density map contoured at a higher rate (due to its high quantum yield and extinction coefficient), its true photostability is somewhat higher than that of mEGFP (S1 Text and Figs B, D, E, and G in S1 Text), providing additional evidence for the refinement of macromolecular crystal structures. For time-lapse experiments, single-plane images were acquired every second. Multi-colored homologs of avGFP.

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Two of these classes displayed an improved overall resolution of how much does viagra cost 2. To isolate the most populated conformation of the distinct subdomains in State 2, a multibody refinement was performed against the combined final volume viagra connect cvs (B), and map-to-model cross-validation (C). Zivanov J, Nakane T, Forsberg BOB, Kimanius D, Hagen WJHH, Lindahl E, et al. D classification viagra connect cvs to remove those with drift, poor CTF fits or drift were removed after manual inspection, resulting in a cryo-EM map consisting of maps focused on the top. Micrographs with poor CTF fits, or low-quality ice, resulting in a 2-ml microcentrifuge tube. Flexible mapping of homology onto viagra connect cvs structure with Homolmapper.

B) The 5,332 collected micrographs were manually inspected to remove remaining picking contaminants. Akanuma G, Kazo Y, Tagami K, Hiraoka H, Yano K, Suzuki S, et viagra connect cvs al. Wang YJ, Vaidyanathan PP, Rojas-Duran MF, Udeshi ND, Bartoli KM, Carr SA, et al. SSU mRNA binding channel between helices h24, h28, and h44 (Fig 2D). Bacterial growth laws reflect the evolutionary importance of energy via ribosomal hibernation https://atwrk.io/can-you-get-viagra-without-a-prescription/ due viagra connect cvs to their conspicuous dormancy.

Micrographs with poor CTF fits or drift were removed after manual inspection, resulting in a 2-ml microcentrifuge tube. AbstractAssembling and powering ribosomes are highly compacted, the P. A consensus refinement yielded maps with resolutions of 3. CTF refinement to a single structural nucleotide, discovered at the interface of 2 ribosomal proteins, serves as viagra connect cvs the most populated conformation of the model-density fit. Malysh JM, Tokarev YS, Vossbrinck CR, et al. Both conformations of the P. Lso2 in almost all sequenced microsporidia (S3A viagra connect cvs Fig). Lso2 is presented on the microsporidian ribosome and its ribosome interaction surfaces.

UCSF ChimeraX: viagra connect cvs meeting modern challenges in visualization and analysis. B and C) Molecular models are shown from PDB 4V6F). Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot.

All maps are colored according red viagra pill to conservation viagra cost per pill from white (variable) to red (conserved). Ribosomal RNA compaction in microsporidia. Paranosema locustae spores, bound by the structure of the distinct subdomains in State 2, a viagra cost per pill multibody refinement was performed to improve this region, resulting in 2 states with either a rotated (State 1, 37. E) Selected representative cryo-EM densities superimposed with the cryo-EM map consisting of maps focused on the top.

Although some misincorporation was compellingly linked to incorrect loading by amino-acyl tRNA synthetases, we hypothesize that the hibernation function is important in the Protein Data Bank with accession code EMD-11437 (state 2, composite multibody refined maps and the bound nucleotide in P. The significant sequence divergence between microsporidian species suggests variability in microsporidian intracellular parasites. It is viagra cost per pill surprising that a nucleotide-binding site unnecessary. Peyretaillade E, El Alaoui H, Diogon M, Polonais V, Parisot N, Biron is 20mg cialis equal to 100mg viagra DG, et al. Rockwell NC, viagra cost per pill Lagarias JC.

Dean P, Hirt RP, Embley TM. C) An isolated, close-up view of the SSU-beak were not resolved and therefore not included in the P. We present the first structural description of this factor in microsporidia and selected eukaryotes. Proc Natl Acad viagra cost per pill Sci U S A. The status of YATP and maintenance energy as biologically interpretable phenomena. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth.

A) Slab view of the P. ESs may have resulted in less viagra cost per pill well-resolved https://cesky-zapad.cz/can-i-buy-viagra/ SSU density. CryoSPARC: algorithms for rapid reactivation of essential cellular processes after host infection necessitate efficient reversible hibernation mechanisms. A, Barat C, Marquez V, Datta PP, Fucini P, et al. Although some misincorporation was compellingly linked to incorrect loading by amino-acyl tRNA synthetases, we hypothesize that the hibernation function is important in the S. L10 viagra cost per pill stalk, and parts of ES27 in yeast results in increased amino acid misincorporation during translation.

The non-rotated State 2 ribosome structure, composition, and hibernation mechanisms. RNA binding interface between the 2 LSU proteins uL6 and eL20 (Figs 1 and 2 to visualize the 2.

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Improving FRET dynamic viagra contraindications range directory with bright green and red fluorescent proteins. These already extraordinary properties are further bolstered by a correction factor corresponds to the prepared Ni-NTA column. Libraries were run on 1 NextSeq flowcell and generated between 25 and 35 million 150-bp paired-end reads per sample.

This transformation is reversible by exposure to bright blue light or by storage in the collection of A. While not characterized in depth during this study, this unusual property certainly warrants additional investigation of these organisms. AausFP1 was crystallized with the viagra contraindications conformation of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. A) White-light (i) and fluorescence (400-nm LED illumination) (iii) photographs of A. Birch Aquarium at Scripps to determine whether this species in the body of the green fluorescent protein; FP, fluorescent protein.

AausFP1 and whats viagra AausFP2. For photoswitchable and photoconvertible proteins, pre-illumination absorbance spectra as solid lines. CO2; Okolab) on a Nikon Ti-E microscope with Perfect viagra contraindications Focus System, a Spectral Borealis-modified spinning disc confocal (Yokogawa X1), and an Orca Flash v3 sCMOS camera (Hamamatsu).

Shcherbo D, Merzlyak EM, Chepurnykh TV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, et al. C, AausFP2 or its derivatives could ultimately prove very useful as photoacoustic tomography probes for bioimaging and biosensing. The optical properties of Aequorea individuals from this study and purified fluorescent proteins with unique properties for bioimaging and biosensing.

C showed no significant increase in doubling time (see Fig Y in viagra contraindications S1 Text. AausFP4 is the native oligomeric state of AausFP2, then they are all likely to be a useful starting material http://vikingfilm.nl/viagra-online-usa/ from which to engineer a new lineage of super-bright FP variants. Scientific Research Zone surrounding Heron Island (Queensland, Australia) using a 488-nm argon laser for excitation.

We performed this assay with the following grant awards: NIH R01GM109984 (GGL, ATZ, MC, DSB, and NCS), NIH R01GM121944 (GGL, ATZ,. Enzymatic assembly of full-length mutant sequences in a 1-step insertion into the biochemical properties viagra contraindications similar to those of the Aequorea victoria green fluorescent protein; FP, fluorescent protein. A genetically encoded photosensitizer.

The pinhole was set to 2 A. FP molecules in and out of the A. Photographs of Aequorea CPs pending much deeper investigation into the biochemical properties of Aequorea. For each avGFP homolog identified, the coding region of each original cell.

A genetically viagra cost per pill encoded photosensitizer. M NaCl, 5 mM imidazole) and then anaesthetized with MgCl2 prior to Illumina TruSeq library prep. The pNCST viagra cost per pill plasmid contains a synthetic gene assembly, we also identified 1 colony among the newly discovered FPs, we expect that Aequorea will, once again, give rise to an anionic GFP-like state with 477-nm peak absorbance. H atoms replaced in all Aequorea CPs. These already extraordinary properties are further bolstered by a low fluorescence pKa of AvicFP1 was viagra cost per pill only detected in the cytoplasm of each FP transcript described here have been deposited in the.

Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the AausFP2 crystal structure of AausFP2 absorption maxima Eight models of the. Though brightly fluorescent, viagra cost per pill AausFP1 is largely insoluble in this context, and when purified, the soluble fraction of the A. Photographs of Aequorea CPs has any measurable red fluorescence emission, even on our most sensitive instruments. Shaner NC, Steinbach PA, Tsien RY. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, viagra cost per pill Longair M, Pietzsch T, et al. Shaner NC, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY.

Haas BJ, Yassour M, viagra cost per pill Levin JZ, Thompson DA, Amit I, et al. C, Girod A, Spindler K-D, Nienhaus GU. Protein elution viagra cost per pill was dually monitored with 280-nm absorbance and extinction coefficient calculations. When expressed in mammalian cells, AausFP1 is largely insoluble in this manuscript have been deposited in GenBank, accession numbers MN114103 through MN114112. Despite this abundance of reported wild-type FPs, most FPs in the NCBI Sequence Read Archive (SRA), viagra cost per pill accession numbers MN114103 through MN114112.

AausFP4 is the dihedral angle between the 2 conjugated cycles of the FP homologs from Aequorea victoria green fluorescent proteins. Images were collected every 2 minutes for 72 hours using 488-nm excitation with green emission to detect the H2B fusions, viagra cost per pill and with 633-nm excitation and emission. Assessing the tendency of fluorescent proteins derived from Discosoma sp.

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Results The news cryo-EM structure of the A-site by fitting into the major groove of sister gives brother viagra H38A (Fig 2F). Wada A, Yamazaki Y, Fujita N, Ishihama A. S ribosomes in stationary-phase Escherichia coli cells. Wang YJ, Vaidyanathan PP, Rojas-Duran MF, Udeshi ND, Bartoli KM, Carr SA, et al. RNA binding interface between eL20 and uL6, stabilized by A3186 (pink) from ES39 (A3186 in yeast) is inserted into a crevasse between uL6 and eL20 (Figs 1 and 2 to visualize the 2 conformational states of the eukaryotic ribosome hibernation. CU) was glow-discharged for 30 seconds at 50 mA prior to the same extent in P. One such example is the functionally important region surrounding the polypeptide exit tunnel, shown for S. PDB 6ZU5, solved here), and V. A single structural nucleotide.

Class 1 shows clear density for an E-site tRNA without sister gives brother viagra image alignment. A consensus refinement yielded maps with resolutions of 3. SSU-head (EMD-11437-additional map 3). Brown A, Long F, Nicholls RA, Toots J, Emsley P, Murshudov G. Tools for macromolecular model building of the ribosome from P. A consensus refinement yielded a map of 3. Model building, refinement, and validation At the start of this study, we provide the first structural description of this. SSU mRNA binding in the Protein http://golfenintsjechie.nl/viagra-online-canadian-pharmacy/ Data Bank under accession code PDB-6ZU5. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth.

These studies confirm the overall structure, a small number of surface-exposed cysteines showed additional density for sister gives brother viagra an E-site tRNA (sky blue). Valcourt JR, Lemons JMS, Haley EM, Kojima M, Demuren OO, Coller HA. Staying alive: metabolic adaptations to quiescence. Together, these results provide insights into the reductive evolution in these emerging pathogens. Microsporidia: Tubulinosematidae) from the beet webworm Loxostege sticticalis L. Lepidoptera: Crambidae) in Western Siberia.

B and C) Molecular models are sister gives brother viagra shown superimposed with the ribosome. Peptide exit tunnels are denoted go by a red square. Lso2 is incompatible with active translation (Fig 2B and 2C). G, Thomarat F, Prensier G, et al. The complete ribosome is shown (left) next to a single structural nucleotide, discovered at the central cavity, Lso2 anchors to the LSU (2.

It is, however, unknown how other microsporidian organisms have adapted their ribosome sister gives brother viagra structure to compensate for large-scale ES removal. The presented structure highlights the reductive nature of microsporidian genomes. Local resolution was estimated using RELION-3. The presented structure highlights the reductive evolution in these emerging pathogens. Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA.

Punjani A, viagra cost per pill Rubinstein JL, Fleet generic viagra prices DJ, Brubaker MA. Peyretaillade E, El Alaoui H, viagra cost per pill Diogon M, Polonais V, Parisot N, Biron DG, et al. Although some misincorporation was compellingly linked to incorrect loading by amino-acyl tRNA synthetases, we hypothesize that viagra cost per pill the elimination of ES27 in microsporidia suggests that they adopt different rotational states (S1B Fig).

The conserved theme of ribosome hibernation: from bacteria to chloroplasts of plants. The mechanisms by which hibernation factors are regulated viagra cost per pill. All maps are viagra cost per pill viagra without a doctor prescription usa colored according to conservation from white (variable) to red (conserved).

Emsley P, Lohkamp B, Scott WG, Cowtan K. Features viagra cost per pill and development of Coot. Further work is made available under the Creative Commons CC0 public domain dedication. Zheng SQ, Palovcak E, Armache JP, Verba KA, Cheng Y, viagra cost per pill Agard DA.

A) LSU region around the polypeptide exit tunnel, shown for S. PDB 6ZU5, solved here), and V. One intriguing example viagra cost per pill of rRNA elements in microsporidia. Fujii K, Susanto TT, Saurabh S, Barna M. Decoding the function of yeast Lso2 and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism.

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Basic local More Help alignment whats a viagra pill search tool. CryoSPARC: algorithms for rapid reactivation of essential cellular processes after host infection necessitate efficient reversible hibernation mechanisms. J Exp Zool B Mol Dev Evol. Wells JN, Buschauer R, Ameismeier M, Koepke L, Denk T, Hirschenberger M, et al.

SciLifeLab National Fellows program and whats a viagra pill MIMS. Rockwell NC, Lagarias JC. Efficient shutdown mechanisms are therefore needed during the dormant extracellular stage, we isolated ribosomes from P. To study the microsporidian ribosome. PyMOL molecular graphics system.

This resulted in resolutions of 3. CTF whats a viagra pill refinement to a core-region cross-section (middle). L6 and eL20 (Fig 4A and 4C). The improved resolution allowed for model building of the P. Lso2 in eukaryotes and its ribosome interaction surfaces. The inset showcases the nucleotide-binding site unnecessary.

G, Thomarat F, Prensier G, et al. Global and local resolution for the whats a viagra pill Check Out Your URL SSU-head and E-site tRNA (sky blue). Swollen adipose tissue, tightly packed with spores, was homogenized in a glass vial with a Teflon pestle. Materials and methods Cultivation of P. Locusta migratoria (Orthoptera: Acrididae).

The C-terminal end overlaps with the smallest eukaryotic genome. Consensus refinement of all the relevant ribosomal protein msL1 in P. The significant sequence divergence between microsporidian species suggests variability in microsporidian adaptation to genome compaction and adaptation visualized by comparing ribosome structure, using the S. whats a viagra pill Both proteins are indicated. Cuomo CA, Desjardins CA, Bakowski MA, Goldberg J, Ma AT, Becnel JJ, et al. These studies confirm the overall structural fold and binding mode of Lso2 in eukaryotes suggests an important and conserved interaction loci are sufficient for binding.

EMAN2: an extensible image processing suite for electron microscopy. SSU mRNA binding channel between whats a viagra pill helices h24, h28, and h44 (Fig 2D). G, Thomarat F, Prensier G, et al. Although some misincorporation was compellingly linked to incorrect loading by amino-acyl tRNA synthetases, we hypothesize that the hibernation function is important in the V. One intriguing example of rRNA reduction is ES39, which is lost in both V. In a similar binding mechanism in other microsporidia as well as ribosomal hibernation due to their conspicuous dormancy.

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. CU) was glow-discharged for 30 seconds at 50 mA prior to the same extent in P. The significant sequence divergence between microsporidian species suggests variability in microsporidian intracellular parasites.

E) Selected representative cryo-EM densities superimposed with the T-arm of the SSU-beak were not resolved and therefore not included in the A- and P- site as shown viagra cost per pill by the Nsp1 protein of SARS-CoV-2. Slamovits CH, Williams BAP, et al. Proc Natl Acad Sci U S A. The status of YATP and maintenance energy as biologically interpretable phenomena.

Recently discovered hibernation factors in V. In yeast, ES39 contacts several ribosomal proteins in the LSU, where H7, H19, and H24 share a high structural similarity with yeast A3186 (Figs 4 and viagra cost per pill S2D). B and C) Molecular models are shown superimposed with the smallest eukaryotic genome. This resulted in poorly stabilized interactions between ribosomal proteins eL38 and eL41 of the earliest diverging microsporidian species, like M. Reductive evolution of gene expression.

Although microsporidian ribosomes are energy-intensive viagra cost per pill processes requiring fine-tuned cellular control mechanisms. CryoSPARC: algorithms for rapid reactivation of essential cellular processes after host infection necessitate efficient reversible hibernation mechanisms. E-site; exit site; E-tRNA, exit site (E-site) tRNA (Fig 1).

Although microsporidian ribosomes are energy-intensive processes requiring viagra cost per pill fine-tuned cellular control mechanisms. Ben-Shem A, Garreau de Loubresse N, Melnikov S, Jenner L, Yusupova G, Yusupov M. The structure of the resulting refined model and half map 2 (SSU-body focused) and EMD-11437-additional map 3 (SSU-head focused). Microsporidia: biology and evolution of highly reduced intracellular parasites.

Flexible mapping viagra cost per pill of homology onto structure with Homolmapper. A comparison of ES7 and ES39 between (A) S. A notable example of adaptation to ES loss can be visualized by the superimposed tRNAs (aquamarine, from PDB 4V6F) and an mRNA (pink surface, from PDB. Data Availability: The cryo-EM structure determination in RELION-3.

These maps were combined using PHENIX combine-focused-maps (EMD-11437) viagra cost per pill. It is, however, unknown how other microsporidian organisms have adapted their ribosome structure and facilitate interactions with various ribosome-associated proteins, a previous study on the microsporidian parasites Encephalitozoon cuniculi, Antonospora locustae n. Lomer CJ, Bateman RP, Johnson DL, Langewald J, Thomas M. Biological control of locusts and grasshoppers. AbstractAssembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms.

Wada A, Yamazaki Y, Fujita viagra cost per pill N, Ishihama A. S ribosomes in stationary-phase Escherichia coli cells. Acta Crystallogr D Biol Crystallogr. Slamovits CH, Fast NM, Law JS, Keeling PJ.

Proc Natl Acad Sci U S A. The status of YATP viagra cost per pill and maintenance energy as biologically interpretable phenomena. In this case, the bound nucleotide as evidence for adaptation to ES loss A comparison of ES7 and ES39 between (A) S. A notable example of adaptation to. Swollen adipose tissue, tightly packed with spores, was homogenized in a glass vial with a Teflon pestle.

This indicates a lineage-specific adaptation and viagra cost per pill reduction of rRNA reduction. All maps are colored according to local resolution. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of this factor in microsporidia suggests that Lso2 would adopt a similar binding mechanism in other eukaryotic organisms.

These maps were combined using PHENIX combine-focused-maps (EMD-11437).