Journal: Science Advances

Article Title: Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages

doi: 10.1126/sciadv.abh0050

Figure Lengend Snippet: ( A and B ) Schematic view of (A) a third instar larval CNS and (B) an adult brain each composed of the ventral nerve cord (vnc) and the brain lobes. The subperineurial and the perineurial glia establish the BBB. The remaining glial cell types are astrocyte-like glia (ALG), cortex glia (CG), wrapping glia (WG), ensheathing glia (EG), and midline glia (MG). The thoracic neuromeres (t1 to t3) expand during pupal development, while the abdominal neuromers a1-8 condense. Some neuropil areas of the adult are indicated. ( C ) Signaling pathways directing the innate immune response. The Toll pathway is preferentially activated by Gram-positive bacteria, and the peptidoglycan recognition proteins (PGRP-LC and PGRP-LE) detect mostly Gram-negative bacteria. All relevant components are indicated. For further details, see main text.

Article Snippet: PGRP-SA , Dm01837989_g1.

Techniques: Protein-Protein interactions, Bacteria

Journal: Science Advances

Article Title: Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages

doi: 10.1126/sciadv.abh0050

Figure Lengend Snippet: Fly stocks.

Article Snippet: PGRP-SA , Dm01837989_g1.

Techniques:

Journal: Science Advances

Article Title: Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages

doi: 10.1126/sciadv.abh0050

Figure Lengend Snippet: TaqMan gene expression assays for qPCR.

Article Snippet: PGRP-SA , Dm01837989_g1.

Techniques: Gene Expression

Journal: Science Advances

Article Title: Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages

doi: 10.1126/sciadv.abh0050

Figure Lengend Snippet: Dissected brains of indicated age stained for Repo (green) to label glial nuclei, HRP (cyan) to label neuronal membranes, and mCherry expression (magenta) directed by the macrophage marker srpHemo-moe::3xmCherry . wL3, wandering third instar larva. Adults were 7 days old. Scale bars, 100 μm. ( A ) Control larva expressing double-stranded RNA directed against green fluorescent protein (GFP). The asterisk indicates six neurons expressing the macrophage marker srpHemo-moe::3xmCherry . ( B ) Control pupa expressing double-stranded RNA directed against GFP. ( C ) Pupal brain expressing PGRP-SA and GNBP1 in glial cells. Note the absence of mCherry-expressing cells in the brain. ( D ) Larval brain expressing PGRP-LC in glial cells, no macrophages are found in the brain. The asterisk denotes neurons weakly expressing the marker srpHemo-moe::3xmCherry . ( E ) PGRP-LC expression is restricted to late larval and pupal stages. Macrophages invade the brain. The dashed line indicates the orthogonal section shown in ( F ). ( G ) Larva with PGRP-LE induction in glial cells. Note the elongated ventral nerve cord. The asterisk indicates neurons weakly expressing the macrophage marker. ( H and J ) Pupal brain expressing PGRP-LE . Note the presence of srpHemo-moe::3xmCherry –expressing cells in the brain. The white dashed line indicates the position of the orthogonal section shown in (J) (arrows indicate macrophages). ( I ) Adult control brain expressing GFP dsRNA . ( K and L ) Adult brain with immunity induction in glia. Cherry-expressing cells are found in the brain. The white dashed line indicates the orthogonal section shown in (L) (arrows indicate macrophages). ( M ) Quantification of the infiltration rate. To count the number of Cherry-positive cells, we used the srpHemo-H2A::3xmCherry marker. Pupae of increasing age were dissected, and the number of Cherry-expressing cells was determined using Imaris. P values are ** P 2–5 hours APF = 0.0034 and **** P 5–12 hours APF < 0.0001 ( t test); n = 10 for every time point.

Article Snippet: PGRP-SA , Dm01837989_g1.

Techniques: Staining, Expressing, Marker, Control

Journal: Science Advances

Article Title: Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages

doi: 10.1126/sciadv.abh0050

Figure Lengend Snippet: ( A , B , D , and E ) Twenty-two to 24-hour-old pupal brains stained as indicated. Glial nuclei (anti-Repo, green), neuronal membranes (anti-HRP, cyan), and invading macrophages (mCherry or dsRed, magenta). Scale bar, 100 μm. (A) Upon concomitant expression of PGRP-LE and silencing of relish expression using RNA interference, no macrophages enter the brain. (B and C ) No suppression of macrophage invasion induced by PGRP-LE expression is observed following concomitant suppression of basket expression. For quantification, see (C) ( n = 6; P = 0.6753, Mann-Whitney). ns, not significant. (D) Pan-glial expression of activated Relish causes an invasion of macrophages into the brain. (E) Neuronal expression of activated Relish is not sufficient to trigger invasion of macrophages into the CNS.

Article Snippet: PGRP-SA , Dm01837989_g1.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: Science Advances

Article Title: Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages

doi: 10.1126/sciadv.abh0050

Figure Lengend Snippet: ( A ) Pan-glial activation of immunity response triggers invasion of many macrophages into the CNS ( n = 10 for repo>>PGRP-LE ; n = 4 for all other genotypes). When pan-glial PGRP-LE expression was blocked in various glial subtypes, the number of invading macrophages is reduced. The color coding of the different glial subtypes is indicated below. For further information about the different Gal4 drivers, see Materials and Methods, . ( B ) Concomitant silencing in cortex and ensheathing glia using nrv2-Gal80 causes a similar reduction in the number of invading macrophages as concomitant silencing in astrocyte-like glial cells using alrm-Gal80 . ( C ) Upon PGRP-LE expression in all glial cells but the BBB, only very few macrophages entered the brain (arrowhead) ( repo-Gal4 , moody-Gal80 , and Tret1-1-Gal80 UAS-PGRP-LE ). ( D ) Average number of invading macrophages in different expression regimes ( n = 10 for repo>>Pvf2; otherwise, n = 4). Color coding is as in (A). In all expression regimes, macrophages enter the brain, except for moodyB4-Gal4 –driven Pvf2 expression. ( E ) Notably, even expression of Pvf2 in only few neurons ( GMR14F11-Gal4 is active in the mushroom bodies only, shown by concomitant expression of UAS-CD8-GFP ), is able to recruit macrophages into the brain lobes [open arrowhead indicates macrophage associated with mushroom body, and filled arrowhead indicates macrophage located in some distance (E and E′)]. Scale bars, 100 μm.

Article Snippet: PGRP-SA , Dm01837989_g1.

Techniques: Activation Assay, Expressing

Journal: Science Advances

Article Title: Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages

doi: 10.1126/sciadv.abh0050

Figure Lengend Snippet: ( A ) The BBB-forming cells are not replaced during onset of pupal development. Confocal image of a 24- to 25-hour APF pupal brain. Larvae with the genotype ( moody-Gal4 UAS-tub-Dendra2 ) were subjected to photoconversion of the subperineurial glia covering the ventral nerve cord. The resultant red fluorescent Dendra2 protein can be detected in pupal brain 25 hours APF. Scale bar, 100 μm. ( B ) Quantification of dye uptake experiments using fluorescein-labeled 70-kDa dextran in control ( repo-Gal4 , UAS-GFP dsRNA ) and after pan-glial immunity induction ( repo-Gal4 , UAS-PGRP-LE ). Datasets were obtained 5 and 24 hours APF. In addition, we included 24-hour APF pupal brains expressing Pvf2 . AU, arbitrary units. OE, overexpression. ( C ) Quantification of changes in fluorescence uptake after 40 min. A slight but significant increase in fluorescein-labeled dextran can be detected following immunity induction (** P = 0.0079) but not following Pvf2 expression ( P = 0.0556). ( D ) Quantification of dye uptake experiments using Texas Red–labeled 10-kDa dextran in control and after pan-glial immunity induction. The same genotypes and time points as in (A) were used. A large variability of data points was found resulting in large error bars. In all genotypes analyzed, an increase in Texas-red–labeled dextran in the CNS can be detected. ( E ) Quantification of changes in Texas-red uptake after 40 min. A significant increase in Texas-red–labeled dextran was found for control brains ( P = 0.0317) and those with a pan-glial immunity induction ( P = 0.0079). However, the levels of Texas-red–labeled dextran were not significantly different between control and pan-glial immunity induction ( P 5hAPF = 0.667 and P 24hAPF = 0.095). Likewise, no differences in Texas-red–labeled dextran uptake were noted in 24-hour APF brains expressing Pvf2 ( P = 0.944). n = 5.

Article Snippet: PGRP-SA , Dm01837989_g1.

Techniques: Labeling, Control, Expressing, Over Expression, Fluorescence

Journal: Science Advances

Article Title: Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages

doi: 10.1126/sciadv.abh0050

Figure Lengend Snippet: ( A ) Longevity of flies with pan-glial immunity induction compared to control flies ( repo-Gal4 , UAS-CD8Cherry versus repo-Gal4 UAS-PGRP-LE ). The viability is markedly reduced ( n = 200 females; P = 3.75 × 10 −92 ). ( B ) Same genotypes as in (A). The climbing ability of 7-day-old flies is markedly reduced upon immunity induction (**** P < 0.0001). ( C to F ) Two-day-old pupae with immunity induction stained for macrophages using [ hml ∆ dsRed ] and subsequent anti-dsRed staining, and either anti-HRP (green) (C and D) to label neuronal membranes or anti-Brp (green) (E and F) to label synapses. Note that macrophages harbor vesicles containing neuronal membrane material [arrowheads in (D′), (F′), and (F″)]. Scale bars, 100 μm (C and E) and 5 μm (D and F).

Article Snippet: PGRP-SA , Dm01837989_g1.

Techniques: Control, Staining, Membrane

Journal: Science Advances

Article Title: Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages

doi: 10.1126/sciadv.abh0050

Figure Lengend Snippet: ( A and B ) Wandering third instar Drosophila larvae injected with GBS were dissected 4 to 5 hours after infection, and the brains were subjected to qPCR. Expression of the genes PGRP-LC , PGRP-SA , Pvf2 , and all AMPs tested ( AttD , DtpB , Def , and Mtk ) was up-regulated upon infection. Mann-Whitney test for Pvf2 , * P = 0.0227; PGRP-LC , ** P = 0.0024; PGRP-SA , *** P = 0.0002; AttD , ** P = 0.0055; DtpB , ** P = 0.0025; Def , * P = 0.0207; Mtk , * P = 0.0499. Unpaired t test for PGRP-LE , P = 0.5297; Tl , P = 0.0988; Dl , P = 0.6202; Dif , P = 0.6758. ( C to E ) Macrophage recruitment and infiltration to GBS-infected pupal brains. Confocal images (top and orthogonal views) showing Drosophila CNS of hml-dsRed pupae 4 to 5 hours after injection in the hemolymph of either (C) Mock or (D and E) GBS. Macrophages ( hml-dsRed in magenta) containing GBS (anti-GBS in green) are detected attached to the CNS (D, inset) or inside the CNS (E). (E′) is a close-up of the dotted boxes from (E). The arrowhead points to a macrophage within the CNS cortex. Phalloidin is in white, and 4′,6-diamidino-2-phenylindole (DAPI) is in blue. Scale bars, 10 μm. ( F ) Quantification of macrophage localization in the whole CNS 4 to 5 hours after injection in the hemolymph of mock (control), GBS, or formaldehyde-fixed GBS. Kruskal-Wallis test followed by Dunn’s multiple comparisons test on brain-associated (attached + entered) macrophages was performed generating adjusted P values: ** P (control vs. GBS) = 0.0011 and P (control vs. fixed-GBS) = 0.7165. Control, n = 21 CNS; GBS, n = 22 CNS; fixed-GBS, n = 10 CNS. ( G ) Number of srpHemo-moe::3xmCherry –expressing macrophages that attach to the brain upon GBS infection of repo-Gal4 animals and repo-Gal4>UAS-relish dsRNA animals [ n = 9; ** P = 0.0078, Mann-Whitney on brain-associated (attached + entered) macrophages].

Article Snippet: PGRP-SA , Dm01837989_g1.

Techniques: Injection, Infection, Expressing, MANN-WHITNEY, Control

Journal: Journal of Hematology & Oncology

Article Title: Off-the-shelf CAR-T cell therapies for relapsed or refractory B-cell malignancies: latest update from ASH 2023 annual meeting

doi: 10.1186/s13045-024-01550-9

Figure Lengend Snippet: Properties of off-the-shelf CAR-T cell therapies updated at ASH 2023

Article Snippet: 1 , ALLO-501/501A , Healthy donor T cells , Cellectis' TALEN® mRNA-mediated TRAC and CD52 KO , CD19 , R/R LBCL and FL , Lentiviral transduction , Phase 1 ALPHA (ALLO-501; NCT03939026) and ALPHA2 (ALLO-501A; NCT04416984) , [ ] .

Techniques: Plasmid Preparation, Transduction, CRISPR, Over Expression, Retroviral

Journal: Scientific Reports

Article Title: Importance of CD8 Tex cell-associated gene signatures in the prognosis and immunology of osteosarcoma

doi: 10.1038/s41598-024-60539-z

Figure Lengend Snippet: Construction of risk prognostic model ( A , B ) UMAP plot of single-cell sequencing in osteosarcoma patients and bar plots ( C ) T-cell differential genes and immune-related genes take intersections. ( D ) Protein–protein interactions interaction network of CD8-Tex-related genes. ( E ) Univariate Cox regression analysis obtained 109 candidate prognostic TRGs for OS.

Article Snippet: Prediction maps were constructed to estimate 1-, 3-, and 5 year survival rates for osteosarcoma patients, including risk scores for CD8 Texcell gene markers and clinicopathologic factors.

Techniques: Sequencing, Protein-Protein interactions

Journal: Current Research in Structural Biology

Article Title: The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

doi: 10.1016/j.crstbi.2021.11.005

Figure Lengend Snippet: Biological function and overall structure of Sa PgsA. ( A ) A cartoon diagram showing the functional role of Sa PgsA in biosynthesis of PG and its derivatives in the membrane. ( B and C ) The overall structure of Sa PgsA dimer shown in cartoon model viewed along membrane plane ( B ) or membrane normal from cytoplasmic side ( C ). Color codes: green, TM1, TM3 and TM5; cyan, TM2, TM4 and TM6; violet, the N-terminal region; lime, the C-terminal region; yellow, loop regions between adjacent TMs; orange, the β-hairpin in the TM1-TM2 loop region; wheat, the short α-helix between TM5 and TM6. The lipid molecules (PGP) are shown as magenta or grey sphere models, whereas the Zinc ions are presented as marine blue spheres. For clarity, one protomer is shown in colored mode, whereas the adjacent protomer is in light grey. ( D – F ) The electrostatic potential surface representation of Sa PgsA. The views are along the membrane plane from two different angles ( D , F ) or along membrane normal from intracellular side ( E ). Color codes: blue, electropositive; white, neutral; red, electronegative. The grey box in the background of B , D and F indicates the approximate position of the hydrophobic region of membrane according to the result of prediction through the Positioning of Proteins in Membrane (PPM) server. The cyan dashed elliptical ring in E indicates the position of active-site cavity within the Sa PgsA monomer, while the black dashed elliptical ring in F labels the approximate location of a lateral portal connecting the active site with lipid bilayer. ( G ) A close-up view of the active site center with a bimetal binding site. The green dashed elliptical ring indicates the location of two metal ions in the active site of Sa PgsA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The gene encoding Sa PgsA was synthesized (GenScript, China) with optimized codon usage for protein expression in E. coli .

Techniques: Functional Assay, Membrane, Binding Assay

Journal: Current Research in Structural Biology

Article Title: The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

doi: 10.1016/j.crstbi.2021.11.005

Figure Lengend Snippet: The PGP binding site in Sa PgsA. ( A ) Interactions between PGP and Sa PgsA through salt bridges and hydrogen bonds. Six evolutionarily conserved residues are colored in green. Black dashed lines indicate the polar interactions between PGP head group and adjacent residues with bond lengths at 2.3–3.0 ​Å. The two blue spheres indicate two metal ions in the active. ( B ) The relative catalytic activity of six point mutants related to the PGP−binding site. The error bars indicate ​± ​SEM with n ​= ​3 or 4. The solid grey bars represent the activity data measured with WT and mutant enzymes solubilized in detergent, whereas the white bars represent the data measured with enzymes embedded in LCP. ( C ) Hydrophobic and van der Waals interactions between the acyl chains of PGP and Sa PgsA. Relevant residues are colored in cyan. The cyan dashed lines indicate the interactions between PGP acyl chains and adjacent residues at distances of 3.2–3.9 ​Å. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The gene encoding Sa PgsA was synthesized (GenScript, China) with optimized codon usage for protein expression in E. coli .

Techniques: Binding Assay, Activity Assay, Mutagenesis

Journal: Current Research in Structural Biology

Article Title: The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

doi: 10.1016/j.crstbi.2021.11.005

Figure Lengend Snippet: The binding site of CDP-DAG in Sa PgsA. ( A ) Interactions of the CDP moiety of CDP-DAG with Sa PgsA. The black dashed lines indicate hydrogen bonds and salt bridges at bond lengths of 2.4 ​Å or 3.1 ​Å. ( B ) Putative CMP release channel (Channel 1) and G3P entrance channel (Channel 2). The PGP molecule from the Sa PgsA−PGP complex structure is superposed onto the Sa PgsA−CDP-DAG complex structure to indicate the putative G3P location. ( C ) Interaction of the fatty acyl chains of CDP-DAG with adjacent amino acid residues in Sa PgsA. The relevant residues are highlighted in cyan. The cyan dashed lines indicate hydrophobic and van der Waals interactions at distance of 3.1–4.0 ​Å. ( D ) The putative entrance for CDP-DAG head group as indicated by the black dashed circle. ( E ) Substrate-dependent kinetic measurement of Sa PgsA activity in detergent micelle. The data points are plotted as mean value ​± ​standard error (SEM as indicated by the error bars. n=3 or 4). For those data points with small errors, the error bars are buried within the symbols. ( F ) The relative catalytic activity of four alanine mutants related to the CDP-DAG−binding site. The error bars indicate ​± ​SEM with n ​= ​3 or 4. The solid grey bars represent the activity data measured with WT and mutant enzymes solubilized in detergent, whereas the white bars represent the data measured with enzymes embedded in LCP. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The gene encoding Sa PgsA was synthesized (GenScript, China) with optimized codon usage for protein expression in E. coli .

Techniques: Binding Assay, Activity Assay, Mutagenesis

Journal: Current Research in Structural Biology

Article Title: The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

doi: 10.1016/j.crstbi.2021.11.005

Figure Lengend Snippet: The bimetal center in Sa PgsA. ( A ) The dependence of PGP synthase activity of Sa PgsA on different divalent metal ions. ( B ) Analysis on the concentration-dependent inhibitory effect of Zn 2+ on Sa PgsA enzymatic activity in detergent micelle. The error bars indicate ​± ​SEM with n ​= ​3 or 4. For those data points with small errors, the error bars are buried within the symbols. ( C and D ) Detailed view of the bimetal binding sites in the Sa PgsA−CDP-DAG complex structure ( C ) and the Sa PgsA−PGP complex structure ( D ). Asp57 and Asp60 on TM2 are depicted as cyan sticks, while Asp78 and Asp82 on TM3 are colored in green. CDP-DAG is shown as light blue stick, whereas PGP is shown as magenta stick. Black dashed lines indicate the coordination bonds at lengths of 2.0–2.3 ​Å. ( E ) The relative catalytic activity of metal-binding site mutants compared to the wild type. In ( A ) and ( E ), the error bars indicate ​± ​SEM with n ​= ​3 or 4. The solid grey bars represent the activity data measured with WT and mutant enzymes solubilized in detergent, whereas the white bars represent the data measured with enzymes embedded in LCP. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The gene encoding Sa PgsA was synthesized (GenScript, China) with optimized codon usage for protein expression in E. coli .

Techniques: Activity Assay, Concentration Assay, Binding Assay, Mutagenesis

Journal: Current Research in Structural Biology

Article Title: The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

doi: 10.1016/j.crstbi.2021.11.005

Figure Lengend Snippet: Structural dynamics of Sa PgsA in response to CDP-DAG or PGP binding. ( A ) Local conformational differences between the structures of Sa PgsA−CDP-DAG complex (white) and the Sa PgsA−PGP complex (green). The blue dashed box indicates conformational changes induced by CDP-DAG binding. The magenta dashed box indicates the conformational changes induced by the binding of PGP head group. PGP and CDP-DAG are presented as stick models in magenta and blue, respectively. ( B ) A zoom-in view of the conformation changes of amino acid residue side chains induced by CDP-DAG binding. The amino acid residues involved in binding the head group of CDP-DAG are presented as stick models in yellow (carbon), blue (nitrogen) and red (oxygen). For comparison, the reference structure ( Sa PgsA−PGP complex) superposed on the one in complex with CDP-DAG is shown in silver. ( C ) An expanded view of the side chain conformation changes induced by PGP binding. The amino acid residues involved in binding the head group of PGP are presented as stick models in green (carbon), blue (nitrogen) and red (oxygen). As a reference, the structure of Sa PgsA−CDP-DAG superposed on the one in complex with PGP is shown in silver. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The gene encoding Sa PgsA was synthesized (GenScript, China) with optimized codon usage for protein expression in E. coli .

Techniques: Binding Assay, Residue, Comparison

Journal: Current Research in Structural Biology

Article Title: The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

doi: 10.1016/j.crstbi.2021.11.005

Figure Lengend Snippet: The daptomycin-resistant point mutants of Sa PgsA. ( A ) The relative enzymatic activity of the daptomycin-resistant point mutants. The error bars indicate ​± ​SEM with n ​= ​3 or 4. The solid grey bars represent the activity data measured with WT and mutant enzymes solubilized in detergent, whereas the white bars represent the data measured with enzymes embedded in LCP. ( B ) Structural mapping of the daptomycin-resistant mutations in Sa PgsA. The residues are colored on the basis of the conservation score of each residue. The amino acid residues subject to mutation for activity assay are shown as sphere models. For clarity, only one protomer of the Sa PgsA dimer is shown.

Article Snippet: The gene encoding Sa PgsA was synthesized (GenScript, China) with optimized codon usage for protein expression in E. coli .

Techniques: Activity Assay, Mutagenesis, Residue

Journal: Current Research in Structural Biology

Article Title: The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

doi: 10.1016/j.crstbi.2021.11.005

Figure Lengend Snippet: A multi-state cartoon model accounting for the catalytic process mediated by Sa PgsA. The grey shading indicates the estimated membrane region. The red and blue elliptical rings indicate the vertical and lateral portals for the entry of G3P and CDP-DAG (or for the release of CMP and PGP) respectively. Among the five putative states (I-V), State II represents the one observed in the Sa PgsA−CDP-DAG complex structure, whereas State V corresponds to the state of the Sa PgsA−PGP complex structure. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The gene encoding Sa PgsA was synthesized (GenScript, China) with optimized codon usage for protein expression in E. coli .

Techniques: Membrane