EMBO/soda-nel huggingface dataset:
Structure and transformation of bacteriophage A511 baseplate and tail upon infection of Listeria cells (2018)
Figure 1

Figure 1. CryoEM reconstructions of the baseplate region of the A511 tail in the pre- and post-host cell attachment states.

A, B, C CryoEM reconstructions of the extended, spontaneously contracted, and urea-contracted tails colored according to the distance to the axis of the reconstruction. The color code bar shown in panel (A) and which is given in Angstroms is the same for all the three panels.

D, E, F The central axial sections of the reconstructions. Note the C-terminal domain of gp104 and other low-density features that do not visualize well in the isosurface representation (panels (A-C)). Letters G, H and I indicate the position of the cross-sections shown in the homonymously labeled panels below.

G A 69 Å-thick slice of the reconstruction of the extended sheath cryoEM map orthogonal to the sixfold axis. The proximal and distal parts of one of the three fibers that run at 82° to the sixfold axis are indicated with a semitransparent cyan and magenta lines, respectively.

F and I Sections of the spontaneously and urea contracted sheath structures that cut through the baseplate/proximal part of the fiber plane, whose position in the reconstructions is indicated with a red line in panels (E and F). The putative direction of the distal part of the fiber that is completely disordered in the reconstructions is indicated with a solar cross symbol.
Structure and transformation of bacteriophage A511 baseplate and tail upon infection of Listeria cells (2018)
Figure 7

Figure 7. Structural transformation of A511 tail upon binding to Listeria cell.

A Percentage of phage particles in the extended, intermediate, and contracted states after 5, 20 and 80 minutes of incubation with L. monocytogenes cell walls. The numbers above the bars show the number of particles contained in the tomograms. The average length of the tail and the standard deviation for each of the three states is given next to the color legend.

B The color code for proteins that were located in A511 cryoEM maps (upper panels C, D, E, F) and their T4 orthologs. The functions are identified with acronyms that are given in Table 2.

C The pre-attachment state. Here and elsewhere in this figure, the EM image in the lower panel is a slice through the cryoET reconstruction. Note that in the presence of the Listeria cell wall the gp108 fibers are no longer in their stowed conformation but instead are fully or partially unraveled.

D Gp108 fibers attach to the host cell wall but the sheath is not yet contracted.

E RBP/TF gp106 (and possibly gp107) pyramids reorient to point toward the cell surface, change their conformation, and bind to the cell wall. The baseplate-proximal part of the sheath is in a contracted state, whereas the capsid-proximal part is still in an extended state. The baseplate-proximal part of the sheath contains a segment where the tail tube is clearly visible because it no longer interacts with the sheath (lower panel).

F The post-attachment state. The tail is fully contracted and the tail tube has pierced through the cell wall. The capsid appears full indicating that DNA injection has not yet begun.

Data information: Scale bar for lower panels (C, D, E, F): 100 nm.

Expanded view figure legends
IFT54 directly interacts with kinesin-II and IFT dynein to regulate anterograde intraflagellar transport (2020)
Figure 1

Figure 1. Two previously undefined and conserved regions of IFT54 are required for proper ciliogenesis.

(A) IFT54 has three previously undefined and conserved regions. Schematic representation of the primary structure of Chlamydomonas IFT54 is shown on the top. Three conserved regions are shown on the bottom. Abbreviations for the species and protein entries: Cr, Chlamydomonas reinhardtii (Cre11.g467739); Hs, Homo sapiens (NP_056465.2); Mm, Mus musculus (NP_082994.1); Ce, Caenorhabditis (NP_508149.1); Dr, Danio, rerio (ABW96885); Dm, Drosophila melanogaster (NP_650353); Tt, Tetrahymena thermophila (XP_001026831.1).

(B) Immunoblot analysis of whole cell samples of the HA-tagged full-length (FL) IFT54, and its deletion mutants as indicated, each expressed in the ift54 null mutant; wild type (WT) and ift54 mutant cells were used as control. Anti-HA was use to visualize the transgenic proteins, antibodies to FLA8/KIF3B were used as a loading control.

(C) Histogram showing ciliary length distribution in populations of ift54 mutant cells transformed with the deletion mutant constructs as indicated. Wild type (WT) cells were used as a control.

(D) Representative DIC images of wild type (WT), ift54 and ift54 cells transformed with the deletion constructs as indicated. Arrows indicate cilia bulges. Scale bar, 5 μm.
IFT54 directly interacts with kinesin-II and IFT dynein to regulate anterograde intraflagellar transport (2020)
Figure 2

Figure 2. IFT-A and IFT-B proteins accumulate at ciliary tip in the IFT54Δ261-275 mutant while at proximal end of cilia in IFT54Δ342-356 mutant.

(A) Immunostaining of IFT54 mutated proteins in various deletion mutants as indicated; wild-type (WT) cells were used as control. Specimens were stained with anti-HA and anti-α-tubulin antibodies followed by imaging using an epifluorescence microscope. Arrows indicate protein accumulation at ciliary tip (left) or proximal end of cilia (right). Scale bars, 5 μm.

(B, C) Immunostaining analysis of IFT43 and IFT38 in the deletion mutants as indicated. Cells were fixed and stained with antibodies against IFT43 (an IFT-A subunit) (B) or against IFT38 (an IFT-B subunit) (C) followed by imaging using both DIC and epifluorescence microscopy. IFT43 and IFT38 accumulated at the ciliary tip in IFT54Δ261-275 mutant but at proximal end of cilia in IFT54Δ342-356 mutant. Arrows indicate ciliary bulges. WT, wild-type cells. Scale bars, 5 μm.
IFT54 directly interacts with kinesin-II and IFT dynein to regulate anterograde intraflagellar transport (2020)
Figure 3

Figure 3. Analysis of ciliary accumulation of IFT motors and IFT complex proteins in IFT54 deletion mutants.

(A) Immunostaining of cells with antibody against IFT dynein subunit D1bLIC. D1bLIC is absent in the cilia bulges at the ciliary tip in IFT54Δ261-275 mutant while enriched at the cilia bulges at proximal end of cilia in IFT54Δ342-356 mutant. WT, wild type cells. Arrows indicate ciliary bulges. Scale bar, 5 μm.

(B) Immunostaining of cells with antibody against kinesin-II subunit FLA10. FLA10 is enriched in cilia bulges at the ciliary tip in IFT54Δ261-275 mutant or at proximal end of cilia in IFT54Δ342-356 mutant. WT, wild type cells. The insets show enhanced and/or enlarged images. Arrows indicate ciliary bulges. Scale bar, 5 μm.

(C, D) Immunoblot analysis of whole cells (C) and isolated cilia (D) of wild type and the three IFT54 deletion mutants. The membranes were probed with antibodies against IFT complex and motor proteins as indicated. Wild-type levels of the various IFT components were detected in the whole cell samples of the deletion strains (C). IFT121, IFT38 and FLA8 accumulated in cilia of both IFT54Δ261-275 and IFT54Δ342-356 mutants. D1bLIC was relatively reduced in IFT54Δ261-275 mutant cilia but was substantially increased in IFT54Δ342-356 mutant cilia (D). Ratios of protein amounts from one representative experiment are in parentheses.
IFT54 directly interacts with kinesin-II and IFT dynein to regulate anterograde intraflagellar transport (2020)
Figure 4

Figure 4. IFT54 interacts with kinesin-II and deletion of residues 342-356 of IFT54 strengthens its interaction with kinesin-II and impairs anterograde IFT.

(A) Kymograms showing the trajectories of IFT trains inside cilia as visualized with IFT43-YFP. An ift43 rescue strain expressing IFT43-YFP (as a control) and the IFT54Δ342-356 mutant expressing IFT43-YFP were analyzed by live imaging via TIRF microscopy.

(B-C) Velocities (B) and frequencies (C) of anterograde and retrograde IFT of IFT43-YFP in control and the IFT54Δ342-356 mutant. n represents the number of cilia assayed from three independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, ***, p<0.0001.

(D) Deletion of residues 342-356 of IFT54 increases its interaction with kinesin-II. Cell extracts from wild type (WT), ift54 expressing HA-tagged full-length (FL) IFT54 or IFT54Δ342-356 mutant were subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. The normalized ratios of FLA8 and FLA10 in the immunoprecipitates (IFT54 versus the mutant) are 1:3.90 and 1:3.13, respectively.

(E) ATP treatment induces dissociation of IFT54 but not IFT54Δ342-356 mutant from kinesin-II. Immunoprecipitation experiments were performed as shown in (D) in the presence or absence of ATP. The normalized ratios of FLA8 and FLA10 in the immunoprecipitates (IFT54 without ATP/with ATP/mutant without ATP/with ATP) are 1:0.37:1.70:1.38 and 1:0.35:1.47:1.45, respectively.

(F) Analysis of the interaction of IFT54 and IFT54Δ342-356 mutant with kinesin-II by GST-pulldown assay. Bacterial expressed GST, GST-tagged IFT54 or IFT54Δ342-356 mutant was mixed respectively with recombinant kinesin-II purified from insect cells in the presence or absence of ATP followed by GST pulldown and immunoblotting. The normalized ratios of KAP, FLA10 and FLA8 in the pull-down samples (GST-IFT54 without ATP/with ATP/mutant without ATP/with ATP) are 1:0.51:1.31:1.44, 1:0.55:1.29:1.19 and 1:0.50:1.31:1.29, respectively.

(G) Interaction of IFT54 with kinesin-II is conserved in mammalian cells. Cell extracts from HEK293T cells expressing eGFP, eGFP tagged TRAF3IP1 or TRAF3IP1Δ535-549 (corresponding deletion mutant of IFT54Δ342-356) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti-GFP and KIF3A antibodies, respectively. Please note, the deletion mutant had an increased interaction with kinesin-II subunit KIF3A relative to the control. The normalized ratio of KIF3A in the immunoprecipitates (TRAF3IP1 versus mutant) is 1:1.76.
IFT54 directly interacts with kinesin-II and IFT dynein to regulate anterograde intraflagellar transport (2020)
Figure 5

Figure 5. IFT54 interacts with IFT dynein subunit D1bLIC and deletion of residues 261-275 of IFT54 disrupts this interaction and impairs anterograde IFT of D1bLIC.

(A) Kymograms showing the trajectories of IFT trains inside cilia as visualized with D1bLIC-YFP. Wild-type (WT) and IFT54Δ261-275 mutant cells expressing D1bLIC-YFP were analyzed by live imaging via TIRF microscopy.

(B, C) Velocities (B) and frequencies (C) of anterograde and retrograde IFT of D1bLIC-YFP. n represents the number of cilia assayed from four independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, n.s.: no significance; ***, p<0.0001.

(D) Kymograms of IFT43-YFP of WT and mutant cilia.

(E, F) Velocities (E) and frequencies (F) of anterograde and retrograde IFT of IFT43-YFP. n represents the number of cilia assayed from three independent experiments. Values show the mean ± S.D. Unpaired two-tailed Student's t test analysis, n.s.: no significance; ***, p<0.0001.

(G) Yeast two hybrid assay for interaction between IFT54 and IFT dynein subunits. Yeast cells that were transformed with each pair of constructs as indicated were grown under selection media lacking leucine, tryptophan, histidine, and adenine (-4) or lacking leucine and tryptophan (-2). DHC1bT, DHC1b tail domain; Empty AD or BD vectors were used as control.

(H) Interaction of IFT54 and IFT54Δ261-275 with D1bLIC by GST-pulldown assay. Bacterial expressed GST, GST-IFT54 or GST-IFT54Δ261-275 were mixed respectively with His-D1bLIC followed by GST pulldown and immunoblotting with anti-GST and anti-His antibodies. The normalized ratio of His-D1bLIC in the pull-down samples (GST-IFT54 versus mutant) is 1:0.32.

(I) Co-immunoprecipitation of IFT54 and IFT54Δ261-275 with D1bLIC. Cell extracts from wild-type (WT), ift54 expressing HA-tagged full-length (FL) IFT54 or IFT54Δ261-275 mutant were subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. Please note, IFT54Δ261-275 mutant exhibited weaker interaction with D1bLIC relative to the control. The normalized ratio of D1bLIC in the immunoprecipitates (IFT54-HA versus mutant) is 1:0.37.

(J) Interaction of IFT54 with D1bLIC is conserved in mammalian cells. Cell extracts from HEK293T cells expressing eGFP, eGFP tagged TRAF3IP1 or TRAF3IP1Δ460-474 (corresponding deletion mutant of IFT54Δ261-275) were analyzed by immunoprecipitation with GFP antibody followed by immunoblotting with anti-GFP and DYNC2LI1, human homologue of D1bLIC, antibodies, respectively. Please note, the deletion mutant weakened the interaction with IFT dynein subunit DYNC2LI1 relative to the control. The normalized ratio of DYNC2LI1 in the immunoprecipitates (TRAF3IP1 versus mutant) is 1:0.49.
Non-canonical metabolic pathways in the malaria parasite detected by isotope-tracing metabolomics (2021)
Figure 4

Figure 4. Inducible disruption of haloacid dehalogenase 4 and a putative Lipin.

A. Anti-HA Western blots for each protein targeted for disruption with a detectable band corresponding to the expected product size. 100 nM rapamycin and 2.5 mM glucosamine were added to induce protein depletion for either one cycle (LDH1) or three cycles (Lipin and HAD4). Anti-PfBiP was used as the loading control.

B. Growth of DiCre-3D7 transgenic lines following LDH1, HAD4, and Lipin depletion with 100 nM rapamycin and 2.5 mM glucosamine was assessed relative to uninduced parasite cultures. The untransfected DiCre-3D7 parasite line was used as a negative control. Data are presented as the mean ± standard error of the mean (SEM) from three independent replicates.

C. Untargeted LC-MS analysis of LDH1 depletion (one cycle of rapamycin and glucosamine treatment) indicated minimal but selective metabolic changes. m/z feature intensities are plotted as the log2 ratio of treated/untreated and the Benjamini-Hochberg corrected P values across six biological replicates plotted as -log10(P). Below, LDH1 substrate and product (pyruvate and lactate respectively) abundance plotted as the ratio of treated/untreated from biological replicates (mean±SEM) and the parental DiCre line presented as the negative control.

D. Loss of Lipin (PF3D7_0303200) leads to accumulation of various lipid species (three cycles of rapamycin and glucosamine treatment). Data are presented as the mean log2 fold change Lipin depleted (treated) enriched trophozoite-stage iRBCs to untreated controls from three to six biological replicates (± SD). The schematic depicts the proposed lipid phosphatase activity of Lipin.

E. Loss of HAD4 leads to increases in intracellular levels of several nucleotides and intermediates in lower glycolysis. Data are represented as the mean log2 fold change of enriched trophozoite-stage iRBCs treated for three cycles compared to untreated controls from three/four biological replicates. Three nucleotide-nucleoside pairs are depicted in the top inset panels, HAD4 disruption leads to accumulation of the nucleotide-monophosphate and depletion of the nucleoside. The schematic depicts lower glycolysis and triose-phosphate interconversion, with the corresponding metabolite levels following HAD4 disruption depicted in the lower right inset.

F. Phosphatase activity against the generic substrate pNPP of wild type HAD4, catalytically inactive HAD4D29A, and a no-enzyme (No Enz.) control, presented as the mean ± SEM (three biological replicates).

G. Inhibition of HAD4 phosphatase activity by inorganic phosphate. Phosphatase activity against the generic substrate pNPP as the mean ± SEM from three independent experiments. The inorganic phosphate IC50 of HAD4 is 2.1 ± 0.21 mM.

H. Substrate specificity of HAD4. Substrates are divided into nucleotide phosphates (black) and other metabolites such as sugar phosphates and vitamins (white). Presented is the enzyme activity from three independent experiments mean ± SEM). Substrate abbreviations are listed in methods.
Non-canonical metabolic pathways in the malaria parasite detected by isotope-tracing metabolomics (2021)
Figure 5

Figure 5. AMR1 depletion leads to impaired isoprenoid biosynthesis, hemoglobin catabolism and growth.

A. Anti-HA detection of AMR1 3'-HA tagged in the presence or absence of 100 nM rapamycin and 2.5 mM glucosamine for three cycles. Anti-PfBiP was used as a loading control.

B. Relative parasitemia was assessed for the untransfected DiCre 3D7 parental line and transfectant parasite lines with AMR1 under inducible disruption. Rapamycin/glucosamine treatment began on cycle zero and parasitemia determined by flow cytometry and compared to identical lines that were left untreated. Data represents the mean relative parasitemia ± SEM from three biological replicates (y-axis) across seven replication cycles (x-axis).

C. Metabolite profile following three cycles of rapamycin and glucosamine treatment. The abundance of isoprenoid biosynthestic intermediates (1-deoxy-xylulose-5-P (DOXP), methyl-erithritol-4-P (MEP), methyl-erithritol-cyclo-2,4-PP (MEcPP), and isopentyl-PP), and hemoglobin-like peptides are presented as the mean log2 fold change from four biological replicates (±SD).
Non-canonical metabolic pathways in the malaria parasite detected by isotope-tracing metabolomics (2021)
Figure 6

Figure 6. The architecture and function of the serine metabolic network.

A. 13C-serine tracing into the metabolic network of iRBC. Black arrows indicate reactions predicted from genomic reconstruction, whereas green arrows indicate reactions that are not predicted but best match the stable-isotope labelling pattern observed. Proposed reactions are annotated below. Metabolites in grey were not observed via mass spectrometry and green indicates a metabolite observed but not predicted. Highlighted in orange is the glycine cleavage system, which is predicted to be active but has not been verified.

B. One-carbon folate metabolism in P. falciparum. The parasite encodes all the enzymes necessary for a complete cytosolic pathway but only appears to possess a single enzyme-serine hydroxymethyltransferase-in the mitochondrion. Highlighted in white are the conventional enzymes and reactions of one-carbon metabolism in the mitochondria of other eukaryotes.

C. HA-tagged SHMT-M detection via immunofluorescence microscopy. DAPI was used to visualise the nucleus, and MitoTracker CMXros used to illuminate the mitochondrion. Anti-HA signal partially colocalises with the mitochondrion.

D. Addition of 100 nM rapamycin and 2.5 mM glucosamine for three growth cycles (+) leads to depletion of the HA-tagged SHMT-M.

E. Depletion of SHMT-M leads to significant changes in intracellular levels of many metabolites in iRBC. m/z features are plotted as the log2 ratio of treated/untreated intensities and the Benjamini-Hochberg corrected P values across six biological replicates plotted as -log10(P).

F. Pathway enrichment analysis of the m/z features identified as significantly different when SHMT-M is depleted. Depicted are the Gene Set Enrichment Analysis (GSEA) P values and Mummichog P values generated via Metaboanalyst 3.0, with the combined P value from each pathway enrichment approach represented by color.
Non-canonical metabolic pathways in the malaria parasite detected by isotope-tracing metabolomics (2021)
Figure 7

Figure 7. SHMT-M is required for mitochondrial function, mETC maintenance and asexual growth.

A. P. falciparum-infected cultures were incubated for 5 hours with 13C15N-isoleucine (heavy). Following protein extraction and trypsin digestion, each isoleucine-containing peptide exists as a heavy (H) and light (L) form, corresponding to newly translated and nascent pools respectively.

B. The log2 H/L ratio reflects the turnover (summation of synthesis and degradation) for each protein detected between the control (untreated) and SHMT-M depleted conditions (thee growth cycles of rapamycin and glucosamine treatment). Data represent the mean log2 H/L ratio across three biological repeats.

C. One-carbon folate intermediates are plotted as the log2 ratio of their abundance detected in treated (100 nM rapamycin / 2.5 mM glucosamine treatment for three growth cycles) compared to untreated in the DiCre parental line (C) and SHMT-M (SM)(mean ±SEM).

D. Pyrimidine biosynthetic intermediates accumulate when SHMT-M is disrupted compared to the DiCre parental line (C) following rapamycin/glucosamine treatment (mean ±SEM). Pyrimidine biosynthetic disruption islinked to dihydroorotate dehydrogenase (DHODH) inhibition and blockage of the mitochondrial electron transport chain (mETC). Ubiquinone (Q) is the necessary electron carrier for the mETC.

E. Asexual growth when SHMT-M is depleted. Parasitemia was determined across seven replication cycles (x-axis) via flow cytometry and presented as the mean ± SEM from three independent experiments. Decylubiquinone (Dub) was added at 10 µM to determine if the growth defect observed was due to impaired ubiquinone synthesis or recycling.
Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis (2020)
Figure 1

Figure 1. Genetic analysis of the mutant pap8-1.

A Structure of the PAP8 locus, blue boxes: exons, lines: introns. Red box: inserted T-DNA as inverted repeats (IR1/IR2) in the first intron. White box with a diagonal red line: deletion at the left border of IR2 and part of second exon (italicized grey sequence). Green and red arrows represent forward and reverse primers respectively as o1: oPAP8_rtp_F ; o2: oPAP8_E3_R; o3: oPAP8_rtp_R; o4:op8i2_R; oLB: oLBb1.3.

B PCR performed on genomic DNA with indicated primers as shown: o1: oPAP8_rtpF, o2: oPAP8_E3R, o3: oPAP8_rtpR, oLB: oLBb1.3, EF1α: ELONGATION FACTOR 1α, WT: wild type, pap8-1: Homozygous albino plant, Ht: Heterozygous green plant; T: T-DNA, arrowhead: 670-bp contaminant amplification product used as loading control.

C RT-PCR on wild type and pap8-1 homozygous plants grown in the dark for 3 days followed with 72-h growth under white light to allow greening of the wild type; EF1α used as control.

D Sequence alignment of predicted full length orthologous PAP8 protein found in representatives of major phylogenetic clades Arabidopsis thaliana, At1g21600; Oryza sativa Indica, EEC67529.1; Amborella trichopoda, XP_006827378.1; Selaginella moellendorffii, XP_002976643.2 Physcomitrella patens, XP_024396032.1. cTP, chloroplast transit peptide as predicted with ChloroP1.1 (www.cbs.dtu.dk) underlined in yellow; HCR shaded in grey, highly conserved region. (*), (:), (.), conserved, strongly similar, or weakly similar amino acid properties (standards from www.uniprot.org). amino acids colors as in Clustal Omega (Red (AVFPMILW): Small + hydrophobic [includes aromomatic - Y]); Blue (DE): Acidic; Magenta (RHK): Basic; Green (STYHCNGQ): Hydroxly + sulfhydryl + amine + G.) bNLS, bipartite NLS as predicted with NLS mapper (http://nls-mapper.iab.keio.ac.jp).

E Half-open siliques of a heterozygous plant showing the embryo greening; scale bar equals 250 µm. The given number is the position rank of the silique from top to bottom of the inflorescence presenting the segregation of homozygous and heterozygous seeds based on their ability to transiently develop chloroplasts.

F Mutant Rescue: WT and two representative pap8-1 plants were grown in vitro using sucrose and low white light intensity (of 10 µmol.m-2.s-1); scale bar equals 20 mm.