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chimallin  (Addgene inc)


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    Addgene inc chimallin
    a , Schematic of the jumbo phage infection cycle. b , Fluorescence microscopy of a 201phi2-1-infected P. chlororaphis cell at 45 mpi ( n = 5 independent experiments). Phage nucleus shell component gp105 (green) is tagged with GFP, phage DNA (blue) is stained with DAPI and the outer cell membrane (red) is stained with FM4-64. c , Tomographic slice of a phage nucleus in a 201phi2-1-infected P. chlororaphis cell at 50–60 mpi. d , Segmentation of the tomogram in c . Outer and inner bacterial membranes are shown in burgundy and pink, respectively. The phage nucleus is coloured blue. Phage capsids and tails are green and cyan, respectively. PhuZ and RecA-like protein filaments are light purple and white, respectively. A subset of 500 host ribosomes is shown in pale yellow. e , Enlarged view of the boxed region in c . Yellow arrows point to the repetitive feature of the phage nucleus perimeter. f , Slice of the cytosolic face of the subtomogram average of the repetitive feature in the phage nucleus perimeter with a comma-shaped subunit outlined in yellow. g , Cytosolic and side views of the shell subtomogram average isosurface with a single subunit outlined in yellow. h , Schematic representation of the p442 -like arrangement of <t>chimallin</t> protomers. A ‘centre’ four-fold symmetry is indicated by a green square and a ‘corner’ four-fold symmetry is indicated by a magenta square. Scale bars: 1 μm ( b ), 250 nm ( c ), 25 nm ( e ) and 10 nm ( f ).
    Chimallin, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chimallin/product/Addgene inc
    Average 90 stars, based on 1 article reviews
    chimallin - by Bioz Stars, 2026-03
    90/100 stars

    Images

    1) Product Images from "Architecture and self-assembly of the jumbo bacteriophage nuclear shell"

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    Journal: Nature

    doi: 10.1038/s41586-022-05013-4

    a , Schematic of the jumbo phage infection cycle. b , Fluorescence microscopy of a 201phi2-1-infected P. chlororaphis cell at 45 mpi ( n = 5 independent experiments). Phage nucleus shell component gp105 (green) is tagged with GFP, phage DNA (blue) is stained with DAPI and the outer cell membrane (red) is stained with FM4-64. c , Tomographic slice of a phage nucleus in a 201phi2-1-infected P. chlororaphis cell at 50–60 mpi. d , Segmentation of the tomogram in c . Outer and inner bacterial membranes are shown in burgundy and pink, respectively. The phage nucleus is coloured blue. Phage capsids and tails are green and cyan, respectively. PhuZ and RecA-like protein filaments are light purple and white, respectively. A subset of 500 host ribosomes is shown in pale yellow. e , Enlarged view of the boxed region in c . Yellow arrows point to the repetitive feature of the phage nucleus perimeter. f , Slice of the cytosolic face of the subtomogram average of the repetitive feature in the phage nucleus perimeter with a comma-shaped subunit outlined in yellow. g , Cytosolic and side views of the shell subtomogram average isosurface with a single subunit outlined in yellow. h , Schematic representation of the p442 -like arrangement of chimallin protomers. A ‘centre’ four-fold symmetry is indicated by a green square and a ‘corner’ four-fold symmetry is indicated by a magenta square. Scale bars: 1 μm ( b ), 250 nm ( c ), 25 nm ( e ) and 10 nm ( f ).
    Figure Legend Snippet: a , Schematic of the jumbo phage infection cycle. b , Fluorescence microscopy of a 201phi2-1-infected P. chlororaphis cell at 45 mpi ( n = 5 independent experiments). Phage nucleus shell component gp105 (green) is tagged with GFP, phage DNA (blue) is stained with DAPI and the outer cell membrane (red) is stained with FM4-64. c , Tomographic slice of a phage nucleus in a 201phi2-1-infected P. chlororaphis cell at 50–60 mpi. d , Segmentation of the tomogram in c . Outer and inner bacterial membranes are shown in burgundy and pink, respectively. The phage nucleus is coloured blue. Phage capsids and tails are green and cyan, respectively. PhuZ and RecA-like protein filaments are light purple and white, respectively. A subset of 500 host ribosomes is shown in pale yellow. e , Enlarged view of the boxed region in c . Yellow arrows point to the repetitive feature of the phage nucleus perimeter. f , Slice of the cytosolic face of the subtomogram average of the repetitive feature in the phage nucleus perimeter with a comma-shaped subunit outlined in yellow. g , Cytosolic and side views of the shell subtomogram average isosurface with a single subunit outlined in yellow. h , Schematic representation of the p442 -like arrangement of chimallin protomers. A ‘centre’ four-fold symmetry is indicated by a green square and a ‘corner’ four-fold symmetry is indicated by a magenta square. Scale bars: 1 μm ( b ), 250 nm ( c ), 25 nm ( e ) and 10 nm ( f ).

    Techniques Used: Infection, Fluorescence, Microscopy, Staining, Membrane

    a , SEC–MALS analysis of purified 201phi2-1 chimallin. The measured molar masses of the three peaks are 6.9 MDa (range 4–13 MDa), 1.2 MDa and 87 kDa (left to right). dRI, differential refractive index. See Extended Data Fig. for molar mass measurements by SEC–MALS. b , c , Z -slices from tomograms of samples from the correspondingly labelled SEC–MALS peaks in a . The full field of view of b is provided in Extended Data Fig. . d , Top left, O -symmetrized reconstruction of the chimallin cubic assembly viewed along the four-fold axis. The protomers of one four-fold face are coloured. Top right, surface representation of the chimallin cubic assembly model viewed along the four-fold axis. Bottom right and bottom left, views of the model along the two-fold and three-fold axes, respectively. Red arrowheads point to the C-terminal segments of the yellow protomer. The green square, pink triangle and black oval indicate that the corresponding panels are viewed down the particle’s four-fold, three-fold and two-fold rotational symmetry axes, respectively. e , Localized asymmetric reconstruction of the chimallin protomer (left) and cartoon model (right). Invading N- and C-terminal segments from neighbouring protomers are coloured blue (NTS), red (CTS1) and burgundy (CTS2). Resolved core protomer termini are shown as spheres. f , Rainbow-coloured cartoon model of the chimallin protomer conformation in the cubic assembly. Resolved N and C termini are shown as spheres. Domains and segments are labelled. Unresolved linkers are shown as dashed lines. g , A rainbow-coloured fold diagram of chimallin (blue at N terminus, red at C terminus) with α-helices labelled alphabetically and β-strands labelled numerically. The N- and C-terminal domains are highlighted in blue and red, respectively. Dashed lines indicate unresolved loops. Scale bars, 50 nm.
    Figure Legend Snippet: a , SEC–MALS analysis of purified 201phi2-1 chimallin. The measured molar masses of the three peaks are 6.9 MDa (range 4–13 MDa), 1.2 MDa and 87 kDa (left to right). dRI, differential refractive index. See Extended Data Fig. for molar mass measurements by SEC–MALS. b , c , Z -slices from tomograms of samples from the correspondingly labelled SEC–MALS peaks in a . The full field of view of b is provided in Extended Data Fig. . d , Top left, O -symmetrized reconstruction of the chimallin cubic assembly viewed along the four-fold axis. The protomers of one four-fold face are coloured. Top right, surface representation of the chimallin cubic assembly model viewed along the four-fold axis. Bottom right and bottom left, views of the model along the two-fold and three-fold axes, respectively. Red arrowheads point to the C-terminal segments of the yellow protomer. The green square, pink triangle and black oval indicate that the corresponding panels are viewed down the particle’s four-fold, three-fold and two-fold rotational symmetry axes, respectively. e , Localized asymmetric reconstruction of the chimallin protomer (left) and cartoon model (right). Invading N- and C-terminal segments from neighbouring protomers are coloured blue (NTS), red (CTS1) and burgundy (CTS2). Resolved core protomer termini are shown as spheres. f , Rainbow-coloured cartoon model of the chimallin protomer conformation in the cubic assembly. Resolved N and C termini are shown as spheres. Domains and segments are labelled. Unresolved linkers are shown as dashed lines. g , A rainbow-coloured fold diagram of chimallin (blue at N terminus, red at C terminus) with α-helices labelled alphabetically and β-strands labelled numerically. The N- and C-terminal domains are highlighted in blue and red, respectively. Dashed lines indicate unresolved loops. Scale bars, 50 nm.

    Techniques Used: Purification, Refractive Index

    Slice through the tomogram of the 201phi2-1 chimallin SEC size-exclusion chromatography void peak. Region marked by a dashed yellow box is used in Figure . Scale bar is 50 nm.
    Figure Legend Snippet: Slice through the tomogram of the 201phi2-1 chimallin SEC size-exclusion chromatography void peak. Region marked by a dashed yellow box is used in Figure . Scale bar is 50 nm.

    Techniques Used: Size-exclusion Chromatography

    a , Relationship of 201phi2-1 chimallin protomer packing in the cube (left) and flat sheet model (right). One protomer is shown as spheres and colored yellow with its NTS in blue and CTS1/CTS2 in red. Protomers that interact directly with this central protomer are colored. Non-interfacing protomers are in white. The flat sheet model is docked within the 201phi2-1 consensus subtomogram average map shown as transparent grey. Red arrows point to locations of unresolved linkers (red dashed lines), and pink symbols indicate 3- or 4-fold symmetry axes. b-d, Close-ups of the 201phi2-1 coordinate model around the binding sites for NTS ( b ), CTS1 ( c ), and CTS2 ( d ). ( e–g ) Close-ups of the Goslar coordinate model around the binding sites for NTS ( e ), CTS1 ( f ), and CTS2 ( g ). For all panels, cryo-EM density map is shown as a mesh at high (pink) and low (grey) contours. Polar interactions are depicted by the symbols indicated in the key at the far right.
    Figure Legend Snippet: a , Relationship of 201phi2-1 chimallin protomer packing in the cube (left) and flat sheet model (right). One protomer is shown as spheres and colored yellow with its NTS in blue and CTS1/CTS2 in red. Protomers that interact directly with this central protomer are colored. Non-interfacing protomers are in white. The flat sheet model is docked within the 201phi2-1 consensus subtomogram average map shown as transparent grey. Red arrows point to locations of unresolved linkers (red dashed lines), and pink symbols indicate 3- or 4-fold symmetry axes. b-d, Close-ups of the 201phi2-1 coordinate model around the binding sites for NTS ( b ), CTS1 ( c ), and CTS2 ( d ). ( e–g ) Close-ups of the Goslar coordinate model around the binding sites for NTS ( e ), CTS1 ( f ), and CTS2 ( g ). For all panels, cryo-EM density map is shown as a mesh at high (pink) and low (grey) contours. Polar interactions are depicted by the symbols indicated in the key at the far right.

    Techniques Used: Binding Assay, Cryo-EM Sample Prep

    a , Topology of the 201phi2-1 chimallin N-terminal domain (NTD, residues 62-228). b , Topology of E. faecalis EF_1977 (PDB ID: 3NAT), the closest structural relative of the chimallin A NTD. The root mean square deviation (RMSD) between chimallin NTD and 3NAT coordinate models is 4.6 Å over 97 aligned Cɑ atoms. Homologous secondary structure elements are colored in yellow. c , Topology of the 201phi2-1 chimallin C-terminal domain (CTD, residues 229-581). d , Topology of the E. coli AtaT tRNA-acetylating toxin (PDB ID: 6AJM) . The root mean square deviation (RMSD) between chimallin CTD and 6AJM coordinate models is 4.2 Å over 269 aligned Cɑ atoms. Homologous secondary structure elements are colored in blue. e , Structural overlay of the chimallin CTD (blue) and AtaT (white; PDB ID 6AJM), showing the similarity in binding site for the chimallin CTS1 segment (red) and the antitoxin AtaR (green).
    Figure Legend Snippet: a , Topology of the 201phi2-1 chimallin N-terminal domain (NTD, residues 62-228). b , Topology of E. faecalis EF_1977 (PDB ID: 3NAT), the closest structural relative of the chimallin A NTD. The root mean square deviation (RMSD) between chimallin NTD and 3NAT coordinate models is 4.6 Å over 97 aligned Cɑ atoms. Homologous secondary structure elements are colored in yellow. c , Topology of the 201phi2-1 chimallin C-terminal domain (CTD, residues 229-581). d , Topology of the E. coli AtaT tRNA-acetylating toxin (PDB ID: 6AJM) . The root mean square deviation (RMSD) between chimallin CTD and 6AJM coordinate models is 4.2 Å over 269 aligned Cɑ atoms. Homologous secondary structure elements are colored in blue. e , Structural overlay of the chimallin CTD (blue) and AtaT (white; PDB ID 6AJM), showing the similarity in binding site for the chimallin CTS1 segment (red) and the antitoxin AtaR (green).

    Techniques Used: Binding Assay

    a , Protomer packing in the cubic 24mer assemblies (left) and flat sheet model (right). One protomer is coloured yellow with NTS in blue and CTS1/CTS2 in red. Protomers interacting directly with this focal protomer are coloured orange, green, blue, purple and red. Non-interfacing protomers are grey. Red dashed lines indicate locations of unresolved linkers, and pink symbols indicate 3- or 4-fold symmetry axes. b , Comparison of chimallin C terminus conformation in the in vitro sheet and the in situ cube. Distances spanned by each disordered segment (CTD–CTS1: residues 582–589 and CTS1–CTS2: residues 612–621) in the two models are noted. c , SEC–MALS of N- and C-terminal truncation mutants (ΔN tail, Δ1–47 (residues 48–631 are present); ΔNTS, Δ1–64 (residues 65–631 are present); ΔCTS2, Δ613–631 (residues 1–611 are present); ΔCTS1+2, Δ583–631 (residues 1–582 are present). See Extended Data Fig. for molar mass measurements by SEC–MALS. d , Relative incorporation of eGFP–chimallin variants into the 201phi2-1 phage nucleus of infected P. chlororaphis cells. Incorporation is calculated as the ratio of GFP fluorescence per pixel in the shell versus outside the shell (details in Extended Data Fig. ). Data are mean ± s.d. Unpaired t -test between a given variant and full-length (FL) ( n = 67 cells); *** P < 0.0001; ΔN tail: n = 51, P = 0.4131; ΔNTS : n = 53, P < 0.0001; ΔCTS1 (residues 1–611): n = 54, P < 0.0001; ΔCTS1+2: n = 50, P = 0.1884; ΔN tail, ΔCTS2 (residues 48–611): n = 58, P < 0.0001; ΔNTS, ΔCTS1+2 (residues 65–582): n = 63, P < 0.0001; eGFP: n = 60, P < 0.0001. The threshold for significance was Bonferroni-corrected to P < 0.007 to account for multiple hypothesis testing.
    Figure Legend Snippet: a , Protomer packing in the cubic 24mer assemblies (left) and flat sheet model (right). One protomer is coloured yellow with NTS in blue and CTS1/CTS2 in red. Protomers interacting directly with this focal protomer are coloured orange, green, blue, purple and red. Non-interfacing protomers are grey. Red dashed lines indicate locations of unresolved linkers, and pink symbols indicate 3- or 4-fold symmetry axes. b , Comparison of chimallin C terminus conformation in the in vitro sheet and the in situ cube. Distances spanned by each disordered segment (CTD–CTS1: residues 582–589 and CTS1–CTS2: residues 612–621) in the two models are noted. c , SEC–MALS of N- and C-terminal truncation mutants (ΔN tail, Δ1–47 (residues 48–631 are present); ΔNTS, Δ1–64 (residues 65–631 are present); ΔCTS2, Δ613–631 (residues 1–611 are present); ΔCTS1+2, Δ583–631 (residues 1–582 are present). See Extended Data Fig. for molar mass measurements by SEC–MALS. d , Relative incorporation of eGFP–chimallin variants into the 201phi2-1 phage nucleus of infected P. chlororaphis cells. Incorporation is calculated as the ratio of GFP fluorescence per pixel in the shell versus outside the shell (details in Extended Data Fig. ). Data are mean ± s.d. Unpaired t -test between a given variant and full-length (FL) ( n = 67 cells); *** P < 0.0001; ΔN tail: n = 51, P = 0.4131; ΔNTS : n = 53, P < 0.0001; ΔCTS1 (residues 1–611): n = 54, P < 0.0001; ΔCTS1+2: n = 50, P = 0.1884; ΔN tail, ΔCTS2 (residues 48–611): n = 58, P < 0.0001; ΔNTS, ΔCTS1+2 (residues 65–582): n = 63, P < 0.0001; eGFP: n = 60, P < 0.0001. The threshold for significance was Bonferroni-corrected to P < 0.007 to account for multiple hypothesis testing.

    Techniques Used: Comparison, In Vitro, In Situ, Infection, Fluorescence, Variant Assay

    a , Domain diagram of 201phi2-1 chimallin (top), with truncations tested by SEC-MALS (bottom). b-h , SEC-MALS analysis of full-length 201phi2-1 chimallin ( b ) and truncated constructs lacking the N-tail ( c ), NTS ( d ), CTS2 ( e ), CTS1+CTS2 ( f ), N-tail+CTS2 ( g ), or NTS + CTS1/2 ( h ). For panels b-h, differential refractive index (dRI) shows protein concentration (blue curves), and yellow points indicate measured molecular weight. Average molecular weight for each peak is shown.
    Figure Legend Snippet: a , Domain diagram of 201phi2-1 chimallin (top), with truncations tested by SEC-MALS (bottom). b-h , SEC-MALS analysis of full-length 201phi2-1 chimallin ( b ) and truncated constructs lacking the N-tail ( c ), NTS ( d ), CTS2 ( e ), CTS1+CTS2 ( f ), N-tail+CTS2 ( g ), or NTS + CTS1/2 ( h ). For panels b-h, differential refractive index (dRI) shows protein concentration (blue curves), and yellow points indicate measured molecular weight. Average molecular weight for each peak is shown.

    Techniques Used: Construct, Refractive Index, Protein Concentration, Molecular Weight

    a , Raw microscopy images of representative cells expressing GFP-chimallin and infected with 201phi2-1 60 min post-infection (mpi) showing GFP fluorescence with associated 3D graphs showing normalized GFP fluorescence intensity within these cells from a top and side view. GFPmut1 was expressed without fusion to chimallin as a negative control and shows no incorporation. Growth curves for P. chlororaphis expressing the indicated 201phi2-1 chimallin truncation mutant (or empty vector control) and challenged with either no phage (black line) or increasing multiplicity of infection of 201phi2-1 (color key at the bottom) over a period of 8 h. Dashed grey-line indicates the half of the maximal optical density at 600 nm achieved by the no phage control in each experiment. Curves are the average of four replicates (n = 4) of each condition. Scale bar: a : 1 μm.
    Figure Legend Snippet: a , Raw microscopy images of representative cells expressing GFP-chimallin and infected with 201phi2-1 60 min post-infection (mpi) showing GFP fluorescence with associated 3D graphs showing normalized GFP fluorescence intensity within these cells from a top and side view. GFPmut1 was expressed without fusion to chimallin as a negative control and shows no incorporation. Growth curves for P. chlororaphis expressing the indicated 201phi2-1 chimallin truncation mutant (or empty vector control) and challenged with either no phage (black line) or increasing multiplicity of infection of 201phi2-1 (color key at the bottom) over a period of 8 h. Dashed grey-line indicates the half of the maximal optical density at 600 nm achieved by the no phage control in each experiment. Curves are the average of four replicates (n = 4) of each condition. Scale bar: a : 1 μm.

    Techniques Used: Microscopy, Expressing, Infection, Fluorescence, Negative Control, Mutagenesis, Plasmid Preparation, Control

    a , Surface model of the 3 × 3 chimallin tetramer lattice viewed from the cytosol with the central tetramer coloured. A ‘centre’ four-fold symmetry is indicated by a green square and a ‘corner’ four-fold symmetry is indicated by a magenta square. b , Surface model of the cytosolic and lumenal faces of the chimallin lattice coloured by relative electrostatic potential (blue: positive; red: negative). c , Cytosolic views of the centre four-fold pore cartoon model with pore-facing residues (Supplementary Table ) shown as sticks. The centre pores ( n = 9) have an average volume of 798 ± 81 nm 3 over the course of 300 ns molecular dynamics simulations ( n = 5; Extended Data Fig. ). d , Same as c , for the corner pore. The core pores ( n = 4) have an average volume of 1,429 ± 227 nm 3 over the course of simulations ( n = 5 independent simulations; Extended Data Fig. ).
    Figure Legend Snippet: a , Surface model of the 3 × 3 chimallin tetramer lattice viewed from the cytosol with the central tetramer coloured. A ‘centre’ four-fold symmetry is indicated by a green square and a ‘corner’ four-fold symmetry is indicated by a magenta square. b , Surface model of the cytosolic and lumenal faces of the chimallin lattice coloured by relative electrostatic potential (blue: positive; red: negative). c , Cytosolic views of the centre four-fold pore cartoon model with pore-facing residues (Supplementary Table ) shown as sticks. The centre pores ( n = 9) have an average volume of 798 ± 81 nm 3 over the course of 300 ns molecular dynamics simulations ( n = 5; Extended Data Fig. ). d , Same as c , for the corner pore. The core pores ( n = 4) have an average volume of 1,429 ± 227 nm 3 over the course of simulations ( n = 5 independent simulations; Extended Data Fig. ).

    Techniques Used:

    a , Unrooted phylogenetic tree of chimallin homologues. Homologues are listed as phage and gene product (gp) numbers (see Supplementary Table ). Groups based on proximity are coloured and the host genus is indicated (Scale bar, 0.1 substitutions per position). b , In situ subtomogram reconstruction of the Goslar chimallin shell. A comma-shaped protomer is marked by a yellow dashed outline and cytosolic and lumenal faces are indicated. c , O -symmetrized map of the Goslar chimallin cubic assembly viewed along the four-fold axis. d , Localized asymmetric reconstruction of the Goslar chimallin protomer. Invading N- and C-terminal segments from neighbouring protomers are coloured blue (NTS), red (CTS1) and burgundy (CTS2). e , Superposition of the Goslar (green) and 201phi2-1 (purple) coordinate models for the cube confirmation of the protomers e . Resolved termini are shown as spheres for the protomers in e . The r.m.s.d. is 1.8 Å for the aligned protomers.
    Figure Legend Snippet: a , Unrooted phylogenetic tree of chimallin homologues. Homologues are listed as phage and gene product (gp) numbers (see Supplementary Table ). Groups based on proximity are coloured and the host genus is indicated (Scale bar, 0.1 substitutions per position). b , In situ subtomogram reconstruction of the Goslar chimallin shell. A comma-shaped protomer is marked by a yellow dashed outline and cytosolic and lumenal faces are indicated. c , O -symmetrized map of the Goslar chimallin cubic assembly viewed along the four-fold axis. d , Localized asymmetric reconstruction of the Goslar chimallin protomer. Invading N- and C-terminal segments from neighbouring protomers are coloured blue (NTS), red (CTS1) and burgundy (CTS2). e , Superposition of the Goslar (green) and 201phi2-1 (purple) coordinate models for the cube confirmation of the protomers e . Resolved termini are shown as spheres for the protomers in e . The r.m.s.d. is 1.8 Å for the aligned protomers.

    Techniques Used: In Situ

    a , Size-exclusion coupled to multi-angle light scattering (SEC-MALS) analysis of purified, full-length Goslar chimallin. b , Exemplar micrograph and 2D class averages. c , Schematic of the localized reconstruction workflow. d , C1 reconstruction filtered and colored by local resolution estimates. e , Unsharpened density map views centered on helix B (residues 64-78) at progressive stages of the localized reconstruction process. Final view of the C1 map shown with a fitted coordinate model. f , g , Fourier shell correlation (FSC) curves for the half-maps and against corresponding models at progressive stages of the localized reconstruction process (red, yellow, and blue), histogram of local resolution estimates for the C1 reconstruction (light blue), and the C1 model-vs-map FSC curve (black). Scale bar: b : 50 nm.
    Figure Legend Snippet: a , Size-exclusion coupled to multi-angle light scattering (SEC-MALS) analysis of purified, full-length Goslar chimallin. b , Exemplar micrograph and 2D class averages. c , Schematic of the localized reconstruction workflow. d , C1 reconstruction filtered and colored by local resolution estimates. e , Unsharpened density map views centered on helix B (residues 64-78) at progressive stages of the localized reconstruction process. Final view of the C1 map shown with a fitted coordinate model. f , g , Fourier shell correlation (FSC) curves for the half-maps and against corresponding models at progressive stages of the localized reconstruction process (red, yellow, and blue), histogram of local resolution estimates for the C1 reconstruction (light blue), and the C1 model-vs-map FSC curve (black). Scale bar: b : 50 nm.

    Techniques Used: Multi-Angle Light Scattering, Purification

    a , Tomographic slice of Goslar-infected APEC2248 cell containing a bonafide phage nucleus, as well as an unidentified spherical body (USB). b , Enlarged view of the phage nucleus and USB from the region boxed in a . c , Plot of the apparent maximal diameter distributions for 201phi2-1 (purple) and Goslar (green) USBs with the summary statistics listed. d , Subtomogram average of the USBs picked from the Goslar dataset. Yellow arrow pointing to putative membrane leaflets. slices of USBs from the Goslar dataset. e , Left, model of USBs as the previously proposed pre-shell/nucleus enclosure of the phage DNA . Right, schematic summary of structural models in this work: (i) exclusion of host nucleases by small chimallin pore sizes, (ii) possible extrusion of phage mRNA via these pores, and (iii) implication of additional shell components to enable uptake of specific phage proteins into the phage nucleus. f-h , Gallery of USBs observed in tomograms of 201phi2-1-infected cell populations. i–l , Gallery of USBs observed in tomograms of Goslar-infected cell populations. Scale bars: a : 150 nm, b : 50 nm, d: 10 nm, f–l : 50 nm.
    Figure Legend Snippet: a , Tomographic slice of Goslar-infected APEC2248 cell containing a bonafide phage nucleus, as well as an unidentified spherical body (USB). b , Enlarged view of the phage nucleus and USB from the region boxed in a . c , Plot of the apparent maximal diameter distributions for 201phi2-1 (purple) and Goslar (green) USBs with the summary statistics listed. d , Subtomogram average of the USBs picked from the Goslar dataset. Yellow arrow pointing to putative membrane leaflets. slices of USBs from the Goslar dataset. e , Left, model of USBs as the previously proposed pre-shell/nucleus enclosure of the phage DNA . Right, schematic summary of structural models in this work: (i) exclusion of host nucleases by small chimallin pore sizes, (ii) possible extrusion of phage mRNA via these pores, and (iii) implication of additional shell components to enable uptake of specific phage proteins into the phage nucleus. f-h , Gallery of USBs observed in tomograms of 201phi2-1-infected cell populations. i–l , Gallery of USBs observed in tomograms of Goslar-infected cell populations. Scale bars: a : 150 nm, b : 50 nm, d: 10 nm, f–l : 50 nm.

    Techniques Used: Infection, Membrane



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    a , Schematic of the jumbo phage infection cycle. b , Fluorescence microscopy of a 201phi2-1-infected P. chlororaphis cell at 45 mpi ( n = 5 independent experiments). Phage nucleus shell component gp105 (green) is tagged with GFP, phage DNA (blue) is stained with DAPI and the outer cell membrane (red) is stained with FM4-64. c , Tomographic slice of a phage nucleus in a 201phi2-1-infected P. chlororaphis cell at 50–60 mpi. d , Segmentation of the tomogram in c . Outer and inner bacterial membranes are shown in burgundy and pink, respectively. The phage nucleus is coloured blue. Phage capsids and tails are green and cyan, respectively. PhuZ and RecA-like protein filaments are light purple and white, respectively. A subset of 500 host ribosomes is shown in pale yellow. e , Enlarged view of the boxed region in c . Yellow arrows point to the repetitive feature of the phage nucleus perimeter. f , Slice of the cytosolic face of the subtomogram average of the repetitive feature in the phage nucleus perimeter with a comma-shaped subunit outlined in yellow. g , Cytosolic and side views of the shell subtomogram average isosurface with a single subunit outlined in yellow. h , Schematic representation of the p442 -like arrangement of chimallin protomers. A ‘centre’ four-fold symmetry is indicated by a green square and a ‘corner’ four-fold symmetry is indicated by a magenta square. Scale bars: 1 μm ( b ), 250 nm ( c ), 25 nm ( e ) and 10 nm ( f ).

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , Schematic of the jumbo phage infection cycle. b , Fluorescence microscopy of a 201phi2-1-infected P. chlororaphis cell at 45 mpi ( n = 5 independent experiments). Phage nucleus shell component gp105 (green) is tagged with GFP, phage DNA (blue) is stained with DAPI and the outer cell membrane (red) is stained with FM4-64. c , Tomographic slice of a phage nucleus in a 201phi2-1-infected P. chlororaphis cell at 50–60 mpi. d , Segmentation of the tomogram in c . Outer and inner bacterial membranes are shown in burgundy and pink, respectively. The phage nucleus is coloured blue. Phage capsids and tails are green and cyan, respectively. PhuZ and RecA-like protein filaments are light purple and white, respectively. A subset of 500 host ribosomes is shown in pale yellow. e , Enlarged view of the boxed region in c . Yellow arrows point to the repetitive feature of the phage nucleus perimeter. f , Slice of the cytosolic face of the subtomogram average of the repetitive feature in the phage nucleus perimeter with a comma-shaped subunit outlined in yellow. g , Cytosolic and side views of the shell subtomogram average isosurface with a single subunit outlined in yellow. h , Schematic representation of the p442 -like arrangement of chimallin protomers. A ‘centre’ four-fold symmetry is indicated by a green square and a ‘corner’ four-fold symmetry is indicated by a magenta square. Scale bars: 1 μm ( b ), 250 nm ( c ), 25 nm ( e ) and 10 nm ( f ).

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: Infection, Fluorescence, Microscopy, Staining, Membrane

    a , SEC–MALS analysis of purified 201phi2-1 chimallin. The measured molar masses of the three peaks are 6.9 MDa (range 4–13 MDa), 1.2 MDa and 87 kDa (left to right). dRI, differential refractive index. See Extended Data Fig. for molar mass measurements by SEC–MALS. b , c , Z -slices from tomograms of samples from the correspondingly labelled SEC–MALS peaks in a . The full field of view of b is provided in Extended Data Fig. . d , Top left, O -symmetrized reconstruction of the chimallin cubic assembly viewed along the four-fold axis. The protomers of one four-fold face are coloured. Top right, surface representation of the chimallin cubic assembly model viewed along the four-fold axis. Bottom right and bottom left, views of the model along the two-fold and three-fold axes, respectively. Red arrowheads point to the C-terminal segments of the yellow protomer. The green square, pink triangle and black oval indicate that the corresponding panels are viewed down the particle’s four-fold, three-fold and two-fold rotational symmetry axes, respectively. e , Localized asymmetric reconstruction of the chimallin protomer (left) and cartoon model (right). Invading N- and C-terminal segments from neighbouring protomers are coloured blue (NTS), red (CTS1) and burgundy (CTS2). Resolved core protomer termini are shown as spheres. f , Rainbow-coloured cartoon model of the chimallin protomer conformation in the cubic assembly. Resolved N and C termini are shown as spheres. Domains and segments are labelled. Unresolved linkers are shown as dashed lines. g , A rainbow-coloured fold diagram of chimallin (blue at N terminus, red at C terminus) with α-helices labelled alphabetically and β-strands labelled numerically. The N- and C-terminal domains are highlighted in blue and red, respectively. Dashed lines indicate unresolved loops. Scale bars, 50 nm.

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , SEC–MALS analysis of purified 201phi2-1 chimallin. The measured molar masses of the three peaks are 6.9 MDa (range 4–13 MDa), 1.2 MDa and 87 kDa (left to right). dRI, differential refractive index. See Extended Data Fig. for molar mass measurements by SEC–MALS. b , c , Z -slices from tomograms of samples from the correspondingly labelled SEC–MALS peaks in a . The full field of view of b is provided in Extended Data Fig. . d , Top left, O -symmetrized reconstruction of the chimallin cubic assembly viewed along the four-fold axis. The protomers of one four-fold face are coloured. Top right, surface representation of the chimallin cubic assembly model viewed along the four-fold axis. Bottom right and bottom left, views of the model along the two-fold and three-fold axes, respectively. Red arrowheads point to the C-terminal segments of the yellow protomer. The green square, pink triangle and black oval indicate that the corresponding panels are viewed down the particle’s four-fold, three-fold and two-fold rotational symmetry axes, respectively. e , Localized asymmetric reconstruction of the chimallin protomer (left) and cartoon model (right). Invading N- and C-terminal segments from neighbouring protomers are coloured blue (NTS), red (CTS1) and burgundy (CTS2). Resolved core protomer termini are shown as spheres. f , Rainbow-coloured cartoon model of the chimallin protomer conformation in the cubic assembly. Resolved N and C termini are shown as spheres. Domains and segments are labelled. Unresolved linkers are shown as dashed lines. g , A rainbow-coloured fold diagram of chimallin (blue at N terminus, red at C terminus) with α-helices labelled alphabetically and β-strands labelled numerically. The N- and C-terminal domains are highlighted in blue and red, respectively. Dashed lines indicate unresolved loops. Scale bars, 50 nm.

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: Purification, Refractive Index

    Slice through the tomogram of the 201phi2-1 chimallin SEC size-exclusion chromatography void peak. Region marked by a dashed yellow box is used in Figure . Scale bar is 50 nm.

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: Slice through the tomogram of the 201phi2-1 chimallin SEC size-exclusion chromatography void peak. Region marked by a dashed yellow box is used in Figure . Scale bar is 50 nm.

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: Size-exclusion Chromatography

    a , Relationship of 201phi2-1 chimallin protomer packing in the cube (left) and flat sheet model (right). One protomer is shown as spheres and colored yellow with its NTS in blue and CTS1/CTS2 in red. Protomers that interact directly with this central protomer are colored. Non-interfacing protomers are in white. The flat sheet model is docked within the 201phi2-1 consensus subtomogram average map shown as transparent grey. Red arrows point to locations of unresolved linkers (red dashed lines), and pink symbols indicate 3- or 4-fold symmetry axes. b-d, Close-ups of the 201phi2-1 coordinate model around the binding sites for NTS ( b ), CTS1 ( c ), and CTS2 ( d ). ( e–g ) Close-ups of the Goslar coordinate model around the binding sites for NTS ( e ), CTS1 ( f ), and CTS2 ( g ). For all panels, cryo-EM density map is shown as a mesh at high (pink) and low (grey) contours. Polar interactions are depicted by the symbols indicated in the key at the far right.

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , Relationship of 201phi2-1 chimallin protomer packing in the cube (left) and flat sheet model (right). One protomer is shown as spheres and colored yellow with its NTS in blue and CTS1/CTS2 in red. Protomers that interact directly with this central protomer are colored. Non-interfacing protomers are in white. The flat sheet model is docked within the 201phi2-1 consensus subtomogram average map shown as transparent grey. Red arrows point to locations of unresolved linkers (red dashed lines), and pink symbols indicate 3- or 4-fold symmetry axes. b-d, Close-ups of the 201phi2-1 coordinate model around the binding sites for NTS ( b ), CTS1 ( c ), and CTS2 ( d ). ( e–g ) Close-ups of the Goslar coordinate model around the binding sites for NTS ( e ), CTS1 ( f ), and CTS2 ( g ). For all panels, cryo-EM density map is shown as a mesh at high (pink) and low (grey) contours. Polar interactions are depicted by the symbols indicated in the key at the far right.

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: Binding Assay, Cryo-EM Sample Prep

    a , Topology of the 201phi2-1 chimallin N-terminal domain (NTD, residues 62-228). b , Topology of E. faecalis EF_1977 (PDB ID: 3NAT), the closest structural relative of the chimallin A NTD. The root mean square deviation (RMSD) between chimallin NTD and 3NAT coordinate models is 4.6 Å over 97 aligned Cɑ atoms. Homologous secondary structure elements are colored in yellow. c , Topology of the 201phi2-1 chimallin C-terminal domain (CTD, residues 229-581). d , Topology of the E. coli AtaT tRNA-acetylating toxin (PDB ID: 6AJM) . The root mean square deviation (RMSD) between chimallin CTD and 6AJM coordinate models is 4.2 Å over 269 aligned Cɑ atoms. Homologous secondary structure elements are colored in blue. e , Structural overlay of the chimallin CTD (blue) and AtaT (white; PDB ID 6AJM), showing the similarity in binding site for the chimallin CTS1 segment (red) and the antitoxin AtaR (green).

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , Topology of the 201phi2-1 chimallin N-terminal domain (NTD, residues 62-228). b , Topology of E. faecalis EF_1977 (PDB ID: 3NAT), the closest structural relative of the chimallin A NTD. The root mean square deviation (RMSD) between chimallin NTD and 3NAT coordinate models is 4.6 Å over 97 aligned Cɑ atoms. Homologous secondary structure elements are colored in yellow. c , Topology of the 201phi2-1 chimallin C-terminal domain (CTD, residues 229-581). d , Topology of the E. coli AtaT tRNA-acetylating toxin (PDB ID: 6AJM) . The root mean square deviation (RMSD) between chimallin CTD and 6AJM coordinate models is 4.2 Å over 269 aligned Cɑ atoms. Homologous secondary structure elements are colored in blue. e , Structural overlay of the chimallin CTD (blue) and AtaT (white; PDB ID 6AJM), showing the similarity in binding site for the chimallin CTS1 segment (red) and the antitoxin AtaR (green).

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: Binding Assay

    a , Protomer packing in the cubic 24mer assemblies (left) and flat sheet model (right). One protomer is coloured yellow with NTS in blue and CTS1/CTS2 in red. Protomers interacting directly with this focal protomer are coloured orange, green, blue, purple and red. Non-interfacing protomers are grey. Red dashed lines indicate locations of unresolved linkers, and pink symbols indicate 3- or 4-fold symmetry axes. b , Comparison of chimallin C terminus conformation in the in vitro sheet and the in situ cube. Distances spanned by each disordered segment (CTD–CTS1: residues 582–589 and CTS1–CTS2: residues 612–621) in the two models are noted. c , SEC–MALS of N- and C-terminal truncation mutants (ΔN tail, Δ1–47 (residues 48–631 are present); ΔNTS, Δ1–64 (residues 65–631 are present); ΔCTS2, Δ613–631 (residues 1–611 are present); ΔCTS1+2, Δ583–631 (residues 1–582 are present). See Extended Data Fig. for molar mass measurements by SEC–MALS. d , Relative incorporation of eGFP–chimallin variants into the 201phi2-1 phage nucleus of infected P. chlororaphis cells. Incorporation is calculated as the ratio of GFP fluorescence per pixel in the shell versus outside the shell (details in Extended Data Fig. ). Data are mean ± s.d. Unpaired t -test between a given variant and full-length (FL) ( n = 67 cells); *** P < 0.0001; ΔN tail: n = 51, P = 0.4131; ΔNTS : n = 53, P < 0.0001; ΔCTS1 (residues 1–611): n = 54, P < 0.0001; ΔCTS1+2: n = 50, P = 0.1884; ΔN tail, ΔCTS2 (residues 48–611): n = 58, P < 0.0001; ΔNTS, ΔCTS1+2 (residues 65–582): n = 63, P < 0.0001; eGFP: n = 60, P < 0.0001. The threshold for significance was Bonferroni-corrected to P < 0.007 to account for multiple hypothesis testing.

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , Protomer packing in the cubic 24mer assemblies (left) and flat sheet model (right). One protomer is coloured yellow with NTS in blue and CTS1/CTS2 in red. Protomers interacting directly with this focal protomer are coloured orange, green, blue, purple and red. Non-interfacing protomers are grey. Red dashed lines indicate locations of unresolved linkers, and pink symbols indicate 3- or 4-fold symmetry axes. b , Comparison of chimallin C terminus conformation in the in vitro sheet and the in situ cube. Distances spanned by each disordered segment (CTD–CTS1: residues 582–589 and CTS1–CTS2: residues 612–621) in the two models are noted. c , SEC–MALS of N- and C-terminal truncation mutants (ΔN tail, Δ1–47 (residues 48–631 are present); ΔNTS, Δ1–64 (residues 65–631 are present); ΔCTS2, Δ613–631 (residues 1–611 are present); ΔCTS1+2, Δ583–631 (residues 1–582 are present). See Extended Data Fig. for molar mass measurements by SEC–MALS. d , Relative incorporation of eGFP–chimallin variants into the 201phi2-1 phage nucleus of infected P. chlororaphis cells. Incorporation is calculated as the ratio of GFP fluorescence per pixel in the shell versus outside the shell (details in Extended Data Fig. ). Data are mean ± s.d. Unpaired t -test between a given variant and full-length (FL) ( n = 67 cells); *** P < 0.0001; ΔN tail: n = 51, P = 0.4131; ΔNTS : n = 53, P < 0.0001; ΔCTS1 (residues 1–611): n = 54, P < 0.0001; ΔCTS1+2: n = 50, P = 0.1884; ΔN tail, ΔCTS2 (residues 48–611): n = 58, P < 0.0001; ΔNTS, ΔCTS1+2 (residues 65–582): n = 63, P < 0.0001; eGFP: n = 60, P < 0.0001. The threshold for significance was Bonferroni-corrected to P < 0.007 to account for multiple hypothesis testing.

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: Comparison, In Vitro, In Situ, Infection, Fluorescence, Variant Assay

    a , Domain diagram of 201phi2-1 chimallin (top), with truncations tested by SEC-MALS (bottom). b-h , SEC-MALS analysis of full-length 201phi2-1 chimallin ( b ) and truncated constructs lacking the N-tail ( c ), NTS ( d ), CTS2 ( e ), CTS1+CTS2 ( f ), N-tail+CTS2 ( g ), or NTS + CTS1/2 ( h ). For panels b-h, differential refractive index (dRI) shows protein concentration (blue curves), and yellow points indicate measured molecular weight. Average molecular weight for each peak is shown.

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , Domain diagram of 201phi2-1 chimallin (top), with truncations tested by SEC-MALS (bottom). b-h , SEC-MALS analysis of full-length 201phi2-1 chimallin ( b ) and truncated constructs lacking the N-tail ( c ), NTS ( d ), CTS2 ( e ), CTS1+CTS2 ( f ), N-tail+CTS2 ( g ), or NTS + CTS1/2 ( h ). For panels b-h, differential refractive index (dRI) shows protein concentration (blue curves), and yellow points indicate measured molecular weight. Average molecular weight for each peak is shown.

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: Construct, Refractive Index, Protein Concentration, Molecular Weight

    a , Raw microscopy images of representative cells expressing GFP-chimallin and infected with 201phi2-1 60 min post-infection (mpi) showing GFP fluorescence with associated 3D graphs showing normalized GFP fluorescence intensity within these cells from a top and side view. GFPmut1 was expressed without fusion to chimallin as a negative control and shows no incorporation. Growth curves for P. chlororaphis expressing the indicated 201phi2-1 chimallin truncation mutant (or empty vector control) and challenged with either no phage (black line) or increasing multiplicity of infection of 201phi2-1 (color key at the bottom) over a period of 8 h. Dashed grey-line indicates the half of the maximal optical density at 600 nm achieved by the no phage control in each experiment. Curves are the average of four replicates (n = 4) of each condition. Scale bar: a : 1 μm.

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , Raw microscopy images of representative cells expressing GFP-chimallin and infected with 201phi2-1 60 min post-infection (mpi) showing GFP fluorescence with associated 3D graphs showing normalized GFP fluorescence intensity within these cells from a top and side view. GFPmut1 was expressed without fusion to chimallin as a negative control and shows no incorporation. Growth curves for P. chlororaphis expressing the indicated 201phi2-1 chimallin truncation mutant (or empty vector control) and challenged with either no phage (black line) or increasing multiplicity of infection of 201phi2-1 (color key at the bottom) over a period of 8 h. Dashed grey-line indicates the half of the maximal optical density at 600 nm achieved by the no phage control in each experiment. Curves are the average of four replicates (n = 4) of each condition. Scale bar: a : 1 μm.

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: Microscopy, Expressing, Infection, Fluorescence, Negative Control, Mutagenesis, Plasmid Preparation, Control

    a , Surface model of the 3 × 3 chimallin tetramer lattice viewed from the cytosol with the central tetramer coloured. A ‘centre’ four-fold symmetry is indicated by a green square and a ‘corner’ four-fold symmetry is indicated by a magenta square. b , Surface model of the cytosolic and lumenal faces of the chimallin lattice coloured by relative electrostatic potential (blue: positive; red: negative). c , Cytosolic views of the centre four-fold pore cartoon model with pore-facing residues (Supplementary Table ) shown as sticks. The centre pores ( n = 9) have an average volume of 798 ± 81 nm 3 over the course of 300 ns molecular dynamics simulations ( n = 5; Extended Data Fig. ). d , Same as c , for the corner pore. The core pores ( n = 4) have an average volume of 1,429 ± 227 nm 3 over the course of simulations ( n = 5 independent simulations; Extended Data Fig. ).

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , Surface model of the 3 × 3 chimallin tetramer lattice viewed from the cytosol with the central tetramer coloured. A ‘centre’ four-fold symmetry is indicated by a green square and a ‘corner’ four-fold symmetry is indicated by a magenta square. b , Surface model of the cytosolic and lumenal faces of the chimallin lattice coloured by relative electrostatic potential (blue: positive; red: negative). c , Cytosolic views of the centre four-fold pore cartoon model with pore-facing residues (Supplementary Table ) shown as sticks. The centre pores ( n = 9) have an average volume of 798 ± 81 nm 3 over the course of 300 ns molecular dynamics simulations ( n = 5; Extended Data Fig. ). d , Same as c , for the corner pore. The core pores ( n = 4) have an average volume of 1,429 ± 227 nm 3 over the course of simulations ( n = 5 independent simulations; Extended Data Fig. ).

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques:

    a , Unrooted phylogenetic tree of chimallin homologues. Homologues are listed as phage and gene product (gp) numbers (see Supplementary Table ). Groups based on proximity are coloured and the host genus is indicated (Scale bar, 0.1 substitutions per position). b , In situ subtomogram reconstruction of the Goslar chimallin shell. A comma-shaped protomer is marked by a yellow dashed outline and cytosolic and lumenal faces are indicated. c , O -symmetrized map of the Goslar chimallin cubic assembly viewed along the four-fold axis. d , Localized asymmetric reconstruction of the Goslar chimallin protomer. Invading N- and C-terminal segments from neighbouring protomers are coloured blue (NTS), red (CTS1) and burgundy (CTS2). e , Superposition of the Goslar (green) and 201phi2-1 (purple) coordinate models for the cube confirmation of the protomers e . Resolved termini are shown as spheres for the protomers in e . The r.m.s.d. is 1.8 Å for the aligned protomers.

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , Unrooted phylogenetic tree of chimallin homologues. Homologues are listed as phage and gene product (gp) numbers (see Supplementary Table ). Groups based on proximity are coloured and the host genus is indicated (Scale bar, 0.1 substitutions per position). b , In situ subtomogram reconstruction of the Goslar chimallin shell. A comma-shaped protomer is marked by a yellow dashed outline and cytosolic and lumenal faces are indicated. c , O -symmetrized map of the Goslar chimallin cubic assembly viewed along the four-fold axis. d , Localized asymmetric reconstruction of the Goslar chimallin protomer. Invading N- and C-terminal segments from neighbouring protomers are coloured blue (NTS), red (CTS1) and burgundy (CTS2). e , Superposition of the Goslar (green) and 201phi2-1 (purple) coordinate models for the cube confirmation of the protomers e . Resolved termini are shown as spheres for the protomers in e . The r.m.s.d. is 1.8 Å for the aligned protomers.

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: In Situ

    a , Size-exclusion coupled to multi-angle light scattering (SEC-MALS) analysis of purified, full-length Goslar chimallin. b , Exemplar micrograph and 2D class averages. c , Schematic of the localized reconstruction workflow. d , C1 reconstruction filtered and colored by local resolution estimates. e , Unsharpened density map views centered on helix B (residues 64-78) at progressive stages of the localized reconstruction process. Final view of the C1 map shown with a fitted coordinate model. f , g , Fourier shell correlation (FSC) curves for the half-maps and against corresponding models at progressive stages of the localized reconstruction process (red, yellow, and blue), histogram of local resolution estimates for the C1 reconstruction (light blue), and the C1 model-vs-map FSC curve (black). Scale bar: b : 50 nm.

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , Size-exclusion coupled to multi-angle light scattering (SEC-MALS) analysis of purified, full-length Goslar chimallin. b , Exemplar micrograph and 2D class averages. c , Schematic of the localized reconstruction workflow. d , C1 reconstruction filtered and colored by local resolution estimates. e , Unsharpened density map views centered on helix B (residues 64-78) at progressive stages of the localized reconstruction process. Final view of the C1 map shown with a fitted coordinate model. f , g , Fourier shell correlation (FSC) curves for the half-maps and against corresponding models at progressive stages of the localized reconstruction process (red, yellow, and blue), histogram of local resolution estimates for the C1 reconstruction (light blue), and the C1 model-vs-map FSC curve (black). Scale bar: b : 50 nm.

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: Multi-Angle Light Scattering, Purification

    a , Tomographic slice of Goslar-infected APEC2248 cell containing a bonafide phage nucleus, as well as an unidentified spherical body (USB). b , Enlarged view of the phage nucleus and USB from the region boxed in a . c , Plot of the apparent maximal diameter distributions for 201phi2-1 (purple) and Goslar (green) USBs with the summary statistics listed. d , Subtomogram average of the USBs picked from the Goslar dataset. Yellow arrow pointing to putative membrane leaflets. slices of USBs from the Goslar dataset. e , Left, model of USBs as the previously proposed pre-shell/nucleus enclosure of the phage DNA . Right, schematic summary of structural models in this work: (i) exclusion of host nucleases by small chimallin pore sizes, (ii) possible extrusion of phage mRNA via these pores, and (iii) implication of additional shell components to enable uptake of specific phage proteins into the phage nucleus. f-h , Gallery of USBs observed in tomograms of 201phi2-1-infected cell populations. i–l , Gallery of USBs observed in tomograms of Goslar-infected cell populations. Scale bars: a : 150 nm, b : 50 nm, d: 10 nm, f–l : 50 nm.

    Journal: Nature

    Article Title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

    doi: 10.1038/s41586-022-05013-4

    Figure Lengend Snippet: a , Tomographic slice of Goslar-infected APEC2248 cell containing a bonafide phage nucleus, as well as an unidentified spherical body (USB). b , Enlarged view of the phage nucleus and USB from the region boxed in a . c , Plot of the apparent maximal diameter distributions for 201phi2-1 (purple) and Goslar (green) USBs with the summary statistics listed. d , Subtomogram average of the USBs picked from the Goslar dataset. Yellow arrow pointing to putative membrane leaflets. slices of USBs from the Goslar dataset. e , Left, model of USBs as the previously proposed pre-shell/nucleus enclosure of the phage DNA . Right, schematic summary of structural models in this work: (i) exclusion of host nucleases by small chimallin pore sizes, (ii) possible extrusion of phage mRNA via these pores, and (iii) implication of additional shell components to enable uptake of specific phage proteins into the phage nucleus. f-h , Gallery of USBs observed in tomograms of 201phi2-1-infected cell populations. i–l , Gallery of USBs observed in tomograms of Goslar-infected cell populations. Scale bars: a : 150 nm, b : 50 nm, d: 10 nm, f–l : 50 nm.

    Article Snippet: Full-length Chimallin from bacteriophages 201phi2-1 (gp105; NCBI Accession YP_001956829.1) and Goslar (gp189; NCBI Accession YP_009820873.1) were cloned with an N-terminal TEV protease-cleavable His 6 tag using UC Berkeley Macrolab vector 2-BT (Addgene #29666).

    Techniques: Infection, Membrane