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- EMDB-36192: DltB tetramer in complex with inhibitor m-AMSA -

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Basic information

Entry
Database: EMDB / ID: EMD-36192
TitleDltB tetramer in complex with inhibitor m-AMSA
Map dataThis map is reconstructed from cryo-EM images collected from K3 camera of TITAN KRIOSg3i and is analysised mainly by the cryoSPARC and Relion.
Sample
  • Complex: DltB tetramer in complex with inhibitor m-AMSA.
    • Protein or peptide: Teichoic acid D-alanyltransferase
  • Ligand: N-[4-(acridin-9-ylamino)-3-methoxyphenyl]methanesulfonamide
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: DIACYL GLYCEROLDiglyceride
Keywordschannel / anti-virulence / MBOAT / DltB / MEMBRANE PROTEIN / MEMBRANE PROTEIN-INHIBITOR complex
Function / homologyD-alanyl transfer protein DltB / Alginate O-acetyltransferase AlgI/D-alanyl transfer protein DltB / lipoteichoic acid biosynthetic process / Membrane bound O-acyl transferase, MBOAT / MBOAT, membrane-bound O-acyltransferase family / acyltransferase activity / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / plasma membrane / Teichoic acid D-alanyltransferase
Function and homology information
Biological speciesStreptococcus thermophilus (bacteria) / Streptococcus thermophilus LMG 18311 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsZhang P / Liu Z
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2024
Title: Structural insights into the transporting and catalyzing mechanism of DltB in LTA D-alanylation.
Authors: Pingfeng Zhang / Zheng Liu /
Abstract: DltB, a model member of the Membrane-Bound O-AcylTransferase (MBOAT) superfamily, plays a crucial role in D-alanylation of the lipoteichoic acid (LTA), a significant component of the cell wall of ...DltB, a model member of the Membrane-Bound O-AcylTransferase (MBOAT) superfamily, plays a crucial role in D-alanylation of the lipoteichoic acid (LTA), a significant component of the cell wall of gram-positive bacteria. This process stabilizes the cell wall structure, influences bacterial virulence, and modulates the host immune response. Despite its significance, the role of DltB is not well understood. Through biochemical analysis and cryo-EM imaging, we discover that Streptococcus thermophilus DltB forms a homo-tetramer on the cell membrane. We further visualize DltB in an apo form, in complex with DltC, and in complex with its inhibitor amsacrine (m-AMSA). Each tetramer features a central hole. The C-tunnel of each protomer faces the intratetramer interface and provides access to the periphery membrane. Each protomer binds a DltC without changing the tetrameric organization. A phosphatidylglycerol (PG) molecule in the substrate-binding site may serve as an LTA carrier. The inhibitor m-AMSA bound to the L-tunnel of each protomer blocks the active site. The tetrameric organization of DltB provides a scaffold for catalyzing D-alanyl transfer and regulating the channel opening and closing. Our findings unveil DltB's dual function in the D-alanylation pathway, and provide insight for targeting DltB as a anti-virulence antibiotic.
History
DepositionMay 16, 2023-
Header (metadata) releaseMay 15, 2024-
Map releaseMay 15, 2024-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36192.png

Downloads & links

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Map

FileDownload / File: emd_36192.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis map is reconstructed from cryo-EM images collected from K3 camera of TITAN KRIOSg3i and is analysised mainly by the cryoSPARC and Relion.
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.7863302 - 1.1035136
Average (Standard dev.)0.0016279727 (±0.025187027)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_36192_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_36192_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DltB tetramer in complex with inhibitor m-AMSA.

EntireName: DltB tetramer in complex with inhibitor m-AMSA.
Components
  • Complex: DltB tetramer in complex with inhibitor m-AMSA.
    • Protein or peptide: Teichoic acid D-alanyltransferase
  • Ligand: N-[4-(acridin-9-ylamino)-3-methoxyphenyl]methanesulfonamide
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: DIACYL GLYCEROLDiglyceride

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Supramolecule #1: DltB tetramer in complex with inhibitor m-AMSA.

SupramoleculeName: DltB tetramer in complex with inhibitor m-AMSA. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Streptococcus thermophilus (bacteria) / Strain: LMG 18311
Molecular weightTheoretical: 48 kDa/nm

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Macromolecule #1: Teichoic acid D-alanyltransferase

MacromoleculeName: Teichoic acid D-alanyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
EC number: Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
Source (natural)Organism: Streptococcus thermophilus LMG 18311 (bacteria) / Strain: LMG 18311
Molecular weightTheoretical: 51.780027 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH NYDIPTTENL YFQGSMIDFL KQLPHLEPYG NPFYFIYLGI ALLPIFIGLF FKKRFAIYEC LVSITFIVLA LTGTHASQI LALLFYIVWQ IIWVYSYKRY RSQRDNKWVF YLHSFLVVLP LILVKVEPTI NGTQSLLNFL GISYLTFRAV G MIIEMRDG ...String:
MGSSHHHHHH NYDIPTTENL YFQGSMIDFL KQLPHLEPYG NPFYFIYLGI ALLPIFIGLF FKKRFAIYEC LVSITFIVLA LTGTHASQI LALLFYIVWQ IIWVYSYKRY RSQRDNKWVF YLHSFLVVLP LILVKVEPTI NGTQSLLNFL GISYLTFRAV G MIIEMRDG VLKEFTLGEF LRFMLFMPTF TSGPIDRFKR FNEDYQSIPN RDELLNMLEQ AVKYIMLGFL YKFVLAQIFG SM LLPPLKA QALSQGGIFN LPTLGVMYVY GFDLFFDFAG YSMFALAVSN LMGIKSPINF DKPFISRDMK EFWNRWHMSL SFW FRDFVF MRLVIVLMRN KVFKNRNTTS NVAYIINMMV MGFWHGITWY YIAYGIFHGI GLVINDAWLR KKKTINKDRK KAGL KPLPE NKWTKALGIF ITFNTVMLSF LIFSGFLNDL WFTKK

UniProtKB: Teichoic acid D-alanyltransferase

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Macromolecule #2: N-[4-(acridin-9-ylamino)-3-methoxyphenyl]methanesulfonamide

MacromoleculeName: N-[4-(acridin-9-ylamino)-3-methoxyphenyl]methanesulfonamide
type: ligand / ID: 2 / Number of copies: 4 / Formula: ASW
Molecular weightTheoretical: 393.459 Da
Chemical component information

ChemComp-ASW:
N-[4-(acridin-9-ylamino)-3-methoxyphenyl]methanesulfonamide / antineoplastic*YM / Amsacrine

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Macromolecule #3: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

MacromoleculeName: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
type: ligand / ID: 3 / Number of copies: 8 / Formula: PGT
Molecular weightTheoretical: 751.023 Da
Chemical component information

ChemComp-PGT:
(1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / phospholipid*YM / Phosphatidylglycerol

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Macromolecule #4: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 4 / Number of copies: 40 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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Macromolecule #5: DIACYL GLYCEROL

MacromoleculeName: DIACYL GLYCEROL / type: ligand / ID: 5 / Number of copies: 2 / Formula: DGA
Molecular weightTheoretical: 625.018 Da
Chemical component information

ChemComp-DGA:
DIACYL GLYCEROL / Diglyceride

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration9 mg/mL
BufferpH: 7.5 / Component - Concentration: 25.0 mM / Component - Name: Hepes / Details: 20 mM Hopes-Na, pH7.5, 0.03% DDM
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: After incubation on the grids at 277K under 100% humidity for 10 s, the grids were bloted for 3.0 s and then plunged frozen into liquid ethane cooled by liquid nitrogen using a Vitrobot..
DetailsThe DltB protein was purified in DDM, the tetramer fractions from gel filtration column were concentrated to about 10 mg/ml.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.1 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6buh


Details: Crystal structure of a membrane-bound O-acyltransferase.
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 294021

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8jem:
DltB tetramer in complex with inhibitor m-AMSA

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