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- PDB-8jes: Cryo-EM structure of DltB homo-tetramer -

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Basic information

Entry
Database: PDB / ID: 8jes
TitleCryo-EM structure of DltB homo-tetramer
ComponentsTeichoic acid D-alanyltransferase
KeywordsMEMBRANE PROTEIN / channel / anti-virulence / MBOAT / DltB
Function / homology
Function and homology information


lipoteichoic acid biosynthetic process / acyltransferase activity / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / plasma membrane
Similarity search - Function
D-alanyl transfer protein DltB / Alginate O-acetyltransferase AlgI/D-alanyl transfer protein DltB / : / Membrane bound O-acyl transferase, MBOAT / MBOAT, membrane-bound O-acyltransferase family
Similarity search - Domain/homology
DIACYL GLYCEROL / Chem-PGT / Teichoic acid D-alanyltransferase
Similarity search - Component
Biological speciesStreptococcus thermophilus LMG 18311 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsZhang, P. / Liu, Z.
Funding support1items
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, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 22, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Teichoic acid D-alanyltransferase
B: Teichoic acid D-alanyltransferase
C: Teichoic acid D-alanyltransferase
D: Teichoic acid D-alanyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)230,74847
Polymers207,1204
Non-polymers23,62843
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking, gel filtration, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Teichoic acid D-alanyltransferase


Mass: 51780.027 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus LMG 18311 (bacteria)
Strain: LMG 18311 / Gene: dltB, stu0762 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41
References: UniProt: Q5M4V4, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
#2: Chemical
ChemComp-PGT / (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / PHOSPHATIDYLGLYCEROL / 1-PALMITOYL-2-OLEOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL)](SODIUM SALT)


Mass: 751.023 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C40H79O10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#3: Sugar...
ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 35 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM
#4: Chemical ChemComp-DGA / DIACYL GLYCEROL


Mass: 625.018 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C39H76O5 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: apo DltB tetramer in DDM. / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 48 kDa/nm / Experimental value: YES
Source (natural)Organism: Streptococcus thermophilus (bacteria) / Strain: LMG 18311
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41(DE3)
Buffer solutionpH: 7.5 / Details: 20 mM Hopes-Na, pH7.5, 0.03% DDM
Buffer componentConc.: 25 mM / Name: Hepes
SpecimenConc.: 9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The DltB protein was purified in DDM, the tetramer fractions from gel filtration column were concentrated to about 10 mg/ml.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
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.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1100 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3538544
3D reconstructionResolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128038 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 152.98 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002816003
ELECTRON MICROSCOPYf_angle_d0.516221488
ELECTRON MICROSCOPYf_chiral_restr0.03962458
ELECTRON MICROSCOPYf_plane_restr0.0042379
ELECTRON MICROSCOPYf_dihedral_angle_d14.0955785

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