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- PDB-8j8j: Membrane bound PRTase, C3 symmetry, donor bound -

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Basic information

Entry
Database: PDB / ID: 8j8j
TitleMembrane bound PRTase, C3 symmetry, donor bound
ComponentsDecaprenyl-phosphate phosphoribosyltransferase
KeywordsMEMBRANE PROTEIN / phosphoribose transferase complex
Function / homology
Function and homology information


decaprenyl-phosphate phosphoribosyltransferase / arabinosyltransferase activity / glycolipid biosynthetic process / capsule polysaccharide biosynthetic process / transferase activity, transferring alkyl or aryl (other than methyl) groups / cell wall organization / magnesium ion binding / plasma membrane
Similarity search - Function
Prenyltransferase / UbiA prenyltransferase family / UbiA prenyltransferase superfamily / UbiA prenyltransferase family
Similarity search - Domain/homology
Chem-PGW / Chem-PRP / Decaprenyl-phosphate phosphoribosyltransferase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.76 Å
AuthorsWu, F.Y. / Gao, S. / Zhang, L. / Rao, Z.H.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)SRPG22-003 China
CitationJournal: Nat Microbiol / Year: 2024
Title: Structural analysis of phosphoribosyltransferase-mediated cell wall precursor synthesis in Mycobacterium tuberculosis.
Authors: Shan Gao / Fangyu Wu / Sudagar S Gurcha / Sarah M Batt / Gurdyal S Besra / Zihe Rao / Lu Zhang /
Abstract: In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl ...In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-β-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.
History
DepositionMay 1, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 27, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Apr 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Decaprenyl-phosphate phosphoribosyltransferase
A: Decaprenyl-phosphate phosphoribosyltransferase
B: Decaprenyl-phosphate phosphoribosyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,53712
Polymers98,0473
Non-polymers3,4909
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, samples were determined as a trimer in EM, gel filtration, samples were eluted as a trimer in gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Decaprenyl-phosphate phosphoribosyltransferase / / 5-phospho-alpha-D-ribose-1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase / DPPR synthase


Mass: 32682.355 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Gene: Rv3806c
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: P9WFR5, decaprenyl-phosphate phosphoribosyltransferase
#2: Sugar ChemComp-PRP / 1-O-pyrophosphono-5-O-phosphono-alpha-D-ribofuranose / ALPHA-PHOSPHORIBOSYLPYROPHOSPHORIC ACID / 1-O-pyrophosphono-5-O-phosphono-alpha-D-ribose / 1-O-pyrophosphono-5-O-phosphono-D-ribose / 1-O-pyrophosphono-5-O-phosphono-ribose / Phosphoribosyl pyrophosphate


Type: D-saccharide / Mass: 390.070 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C5H13O14P3 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
a-D-Ribf1PO35PO3IUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-PGW / (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-(1-glycerol)] / PHOSPHATIDYLGLYCEROL / Phosphatidylglycerol


Mass: 749.007 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C40H77O10P / Comment: phospholipid*YM
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: trimeric phosphoribosyltransferase / Type: COMPLEX / Details: trimeric phosphoribosyltransferase, donor bound / Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.0978 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMHydroxyethylpiperazine Ethane Sulfonic AcidHepes1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono disperse
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 9908
EM imaging opticsEnergyfilter name: TFS Selectris

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4particle selectionblob picker was used to automatically select particle images
2EPU2.12image acquisitionautomatic selection
4cryoSPARC4CTF correction
10cryoSPARC4initial Euler assignment
11cryoSPARC4final Euler assignment
12cryoSPARC4classification
13cryoSPARC43D reconstruction
CTF correctionDetails: CTF amplitude correction was performed following 3D reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3597451
3D reconstructionResolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 410532 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0057023
ELECTRON MICROSCOPYf_angle_d0.7399567
ELECTRON MICROSCOPYf_dihedral_angle_d25.3161080
ELECTRON MICROSCOPYf_chiral_restr0.0421128
ELECTRON MICROSCOPYf_plane_restr0.0041143

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