Journal: Nucleic Acids Research

Article Title: Bacterial WYL domain transcriptional repressors sense single-stranded DNA to control gene expression

doi: 10.1093/nar/gkae1101

Figure Lengend Snippet: CapW activates transcription in response to DNA damage. ( A ) Top: Schematic of the E. coli upec-117 CBASS operon. The capW gene is shown in yellow, cdcC (CD-NTase) in orange, cap17 (TPALS-PNP effector) in green and cap7 / cap8 (HORMA and Trip13 regulators) in blue. Putative regulators cap18 and cap19 are shown in gray. The bidirectional promoter is indicated by two arrows. Bottom: Schematic of the capW GFP reporter construct. The intergenic region containing the bidirectional promoter is the same as in the native E. coli upec-117 CBASS operon. ( B ) Western blot showing activation of GFP reporter expression upon treatment of log-phase E. coli cultures with DNA damaging agents mitomycin C (1 μg/ml, left) or zeocin (100 μg/ml, right). α-RNAP: anti-RNA polymerase loading control. ( C ) Top: Schematic of homologous recombination in E. coli . RecBCD resects DNA double-strand breaks to enable strand exchange by RecA. In Δ recA cells, the ssDNA products of resection are expected to accumulate, whereas ssDNA production is limited in Δ recB cells. Bottom: Western blot showing GFP reporter expression in E. coli strains lacking functional RecBCD (Δ recB ) or RecA (Δ recA ), in the absence of DNA damage (left) or 90 min after the addition of zeocin (right). ( D ) Top: Domain schematic of E. coli upec-117 CapW, with positions of WYL domain point mutations highlighted. Bottom: Western blot showing GFP reporter expression driven by CapW WYL domain mutants, in the absence of DNA damage (left) or 90 min after the addition of zeocin (right).

Article Snippet: The wild-type (WT) gene encoding the full-length CapW from Rhizobium leguminosarum (NCBI #WP 033184384.1) was also synthesized (IDT) and cloned into UC Berkeley Macrolab vector 2BT (Addgene #29666).

Techniques: Construct, Western Blot, Activation Assay, Expressing, Control, Homologous Recombination, Functional Assay

Journal: Nucleic Acids Research

Article Title: Bacterial WYL domain transcriptional repressors sense single-stranded DNA to control gene expression

doi: 10.1093/nar/gkae1101

Figure Lengend Snippet: CapW–ssDNA binding inhibits dsDNA binding. ( A ) FP assay showing the binding of a 7mer poly-T ssDNA to E. coli upec-117 CapW (WT, circles; R200A, squares; and S158E, triangles). Data represent the mean ± standard deviation of triplicate samples, fit with a one-site binding model. K d values could not be determined for R200A and S158E mutants. See for CapW binding different 7mer ssDNAs, and for ssDNA versus ssRNA binding. ( B ) FP assay showing the binding of WT E. coli upec-117 CapW to its preferred palindromic binding site (30mer dsDNA), in the absence of ssDNA (solid circles and solid line) or in the presence of 0.5 mM 7mer poly-T ssDNA (open circles and dashed line). See for dsDNA binding in different concentrations of ssDNA. ( C ) FP assay showing the binding of CapW R200A to dsDNA, in the absence of ssDNA (solid squares and solid line) or in the presence of 0.5 mM 7mer poly-T ssDNA (open squares and dashed line). ( D ) FP assay showing the binding of CapW S158E to dsDNA, in the absence of ssDNA (solid triangles and solid line) or in the presence of 0.5 mM 7mer poly-T ssDNA (open triangles and dashed line).

Article Snippet: The wild-type (WT) gene encoding the full-length CapW from Rhizobium leguminosarum (NCBI #WP 033184384.1) was also synthesized (IDT) and cloned into UC Berkeley Macrolab vector 2BT (Addgene #29666).

Techniques: Binding Assay, FP Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Bacterial WYL domain transcriptional repressors sense single-stranded DNA to control gene expression

doi: 10.1093/nar/gkae1101

Figure Lengend Snippet: Structure of a CapW–ssDNA complex. ( A ) Domain schematic of Rl CapW. ( B ) Side view of the ssDNA-bound Rl CapW dimer, with one protomer colored as in panel (A) with individual domains and N- and C-termini labeled; and the second protomer colored gray. ( C ) Top view of the ssDNA-bound Rl CapW dimer. Bound ssDNA is colored black and shown as sticks. Dotted box indicates the area shown in panels (D) and (E). ( D ) Closeup view of ssDNA binding to Rl CapW. Conserved amino acids that contact DNA are shown as sticks. See for Fo – Fc electron density at 3.0 σ from an omit map calculated without modeled DNA. ( E ) View as in panel (D), showing the electrostatic surface of CapW (red indicates negative charge and blue indicates positive charge). ( F ) Sequence alignment of the WYL domains of Rl CapW (IMG # 2631366338), Ec CapW (NCBI #WP_001534693.1), Sm CapW (IMG #2657474953), E. fergusonii BrxR (UniProt ID B7L3Y3) and Acinetobacter sp. NEB 394 BrxR (UniProt ID A0A7H8SL41). Conserved residues that contact ssDNA are marked, and the conserved RWHVR motif is indicated.

Article Snippet: The wild-type (WT) gene encoding the full-length CapW from Rhizobium leguminosarum (NCBI #WP 033184384.1) was also synthesized (IDT) and cloned into UC Berkeley Macrolab vector 2BT (Addgene #29666).

Techniques: Labeling, Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Bacterial WYL domain transcriptional repressors sense single-stranded DNA to control gene expression

doi: 10.1093/nar/gkae1101

Figure Lengend Snippet: ssDNA binding induces conformational changes in CapW. ( A ) Structure of the Sm CapW dimer, with domains colored as in Figure . Bound DNA is modeled from a structure of Acinetobacter BrxR bound to DNA (see ) . ( B ) Overlay of Rl CapW bound to ssDNA (domains colored as in Figure ) and Sm CapW (gray). ( C ) Closeup view of the Rl CapW RWHVR motif (yellow) contacting ssDNA (black), and conformational changes in the WYL (yellow), linker (white) and wHTH domains (blue) that result from ssDNA binding. Equivalent structural elements of Sm CapW are shown in gray.

Article Snippet: The wild-type (WT) gene encoding the full-length CapW from Rhizobium leguminosarum (NCBI #WP 033184384.1) was also synthesized (IDT) and cloned into UC Berkeley Macrolab vector 2BT (Addgene #29666).

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: Bacterial WYL domain transcriptional repressors sense single-stranded DNA to control gene expression

doi: 10.1093/nar/gkae1101

Figure Lengend Snippet: CapW aids CBASS-mediated protection against lysogenic phage induction. ( A ) Schematic of experiment, showing E. coli cells carrying a heat shock-inducible λ prophage and a plasmid carrying either the full E. coli upec-117 CBASS system (for panel B) or a GFP reporter plasmid (for panel C). Cells were heat-shocked to induce the lytic cycle, then the resulting phages were titered (panel B) or GFP reporter expression was measured by western blot (panel C). ( B ) λ phage titer produced by lytic induction in E. coli cells carrying an empty vector (EV), WT E. coli upec-117 CBASS, or CBASS with a catalytically dead CdnC (CdnC C.D.: D73N/D75N) or with CapW R200A or S158E mutations. Significance values of paired t -tests: EV versus WT ( P = 0.0153); EV versus CdnC C.D. (0.9734); EV versus CapW R200A (0.0045); and EV versus CapW S158E (0.0042). See for plaque data. ( C ) Western blot showing activation of GFP reporter expression (α-GFP) controlled by WT CapW after heat shock, in cells either lacking a λ prophage (left) or carrying a λ prophage (right). The lane marked (+) is a positive control for cells expressing GFP. α-RNAP: anti-RNA polymerase loading control. ( D ) Western blot showing activation of GFP reporter expression (α-GFP) controlled by CapW mutants (R200A or S158E) after heat shock, in cells carrying a λ prophage. The lane marked (+) is a positive control for cells expressing GFP. α-RNAP: anti-RNA polymerase loading control.

Article Snippet: The wild-type (WT) gene encoding the full-length CapW from Rhizobium leguminosarum (NCBI #WP 033184384.1) was also synthesized (IDT) and cloned into UC Berkeley Macrolab vector 2BT (Addgene #29666).

Techniques: Plasmid Preparation, Expressing, Western Blot, Produced, Activation Assay, Positive Control, Control

Journal: Nucleic Acids Research

Article Title: Bacterial WYL domain transcriptional repressors sense single-stranded DNA to control gene expression

doi: 10.1093/nar/gkae1101

Figure Lengend Snippet: Model for CapW-mediated CBASS expression. In unperturbed cells, CapW binds the CBASS promoter to repress transcription. Upon DNA damage or another stress signal, lysogenic phages are induced (lower path), which contributes to the production of ssDNA fragments. ssDNA binds to the CapW WYL domains to induce a conformational change that leads to dissociation from the CBASS promoter and increased CBASS expression. In E. coli upec-117 CBASS, the Cap17 effector depletes ATP to arrest growth and potentially cause cell death , preventing phages from escaping the cell.

Article Snippet: The wild-type (WT) gene encoding the full-length CapW from Rhizobium leguminosarum (NCBI #WP 033184384.1) was also synthesized (IDT) and cloned into UC Berkeley Macrolab vector 2BT (Addgene #29666).

Techniques: Expressing

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

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

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

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

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

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

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

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

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:

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

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

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