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- PDB-8if8: Arabinosyltransferase AftA -

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Basic information

Entry
Database: PDB / ID: 8if8
TitleArabinosyltransferase AftA
ComponentsGalactan 5-O-arabinofuranosyltransferase
KeywordsMEMBRANE PROTEIN / Arabinosyltransferase / AftA / Mycobacterium tuberculosis
Function / homology
Function and homology information


galactan 5-O-arabinofuranosyltransferase / cell wall macromolecule biosynthetic process / UDP-galactosyltransferase activity / mycolate cell wall layer assembly / capsule polysaccharide biosynthetic process / glycosyltransferase activity / peptidoglycan-based cell wall / cell wall organization / plasma membrane
Similarity search - Function
Arabinofuranosyltransferase AftA, C-terminal / Arabinofuranosyltransferase AftA, N-terminal / Arabinofuranosyltransferase A C terminal / Arabinofuranosyltransferase N terminal
Similarity search - Domain/homology
Galactan 5-O-arabinofuranosyltransferase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsGong, Y.C. / Rao, Z.H. / Zhang, L.
Funding support China, 1items
OrganizationGrant numberCountry
Other governmentinternal fund China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structure of the priming arabinosyltransferase AftA required for AG biosynthesis of .
Authors: Yicheng Gong / Chuancun Wei / Jun Wang / Nengjiang Mu / Qinhong Lu / Chengyao Wu / Ning Yan / Huifang Yang / Yao Zhao / Xiuna Yang / Sudagar S Gurcha / Natacha Veerapen / Sarah M Batt / ...Authors: Yicheng Gong / Chuancun Wei / Jun Wang / Nengjiang Mu / Qinhong Lu / Chengyao Wu / Ning Yan / Huifang Yang / Yao Zhao / Xiuna Yang / Sudagar S Gurcha / Natacha Veerapen / Sarah M Batt / Zhiqiang Hao / Lintai Da / Gurdyal S Besra / Zihe Rao / Lu Zhang /
Abstract: Arabinogalactan (AG) is an essential cell wall component in mycobacterial species, including the deadly human pathogen . It plays a pivotal role in forming the rigid mycolyl-AG-peptidoglycan core for ...Arabinogalactan (AG) is an essential cell wall component in mycobacterial species, including the deadly human pathogen . It plays a pivotal role in forming the rigid mycolyl-AG-peptidoglycan core for in vitro growth. AftA is a membrane-bound arabinosyltransferase and a key enzyme involved in AG biosynthesis which bridges the assembly of the arabinan chain to the galactan chain. It is known that AftA catalyzes the transfer of the first arabinofuranosyl residue from the donor decaprenyl-monophosphoryl-arabinose to the mature galactan chain (i.e., priming); however, the priming mechanism remains elusive. Herein, we report the cryo-EM structure of AftA. The detergent-embedded AftA assembles as a dimer with an interface maintained by both the transmembrane domain (TMD) and the soluble C-terminal domain (CTD) in the periplasm. The structure shows a conserved glycosyltransferase-C fold and two cavities converging at the active site. A metal ion participates in the interaction of TMD and CTD of each AftA molecule. Structural analyses combined with functional mutagenesis suggests a priming mechanism catalyzed by AftA in AG biosynthesis. Our data further provide a unique perspective into anti-TB drug discovery.
History
DepositionFeb 17, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 7, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Galactan 5-O-arabinofuranosyltransferase
B: Galactan 5-O-arabinofuranosyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,2344
Polymers139,1542
Non-polymers802
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Galactan 5-O-arabinofuranosyltransferase / Arabinofuranosyltransferase AftA


Mass: 69576.938 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Gene: aftA, Rv3792 / Plasmid: PMV261
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain (production host): MC2 155
References: UniProt: P9WN03, galactan 5-O-arabinofuranosyltransferase
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / 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: AftA dimer / Type: COMPLEX / Details: AftA subunit1, AftA subunit2 / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.0695 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Strain: MC2 155
Buffer solutionpH: 8 / Details: 150mM NaCl, 20mM Hepes, 0.04%GDN
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaCl
SpecimenConc.: 6.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: the sample was mono disperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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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 FIELD / Nominal magnification: 10500 X / Calibrated magnification: 59000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.4 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 6113 / Num. of real images: 6113 / Details: images were collected in high resolution mode
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2752481
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 147896 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0069553
ELECTRON MICROSCOPYf_angle_d0.73513073
ELECTRON MICROSCOPYf_dihedral_angle_d11.9831345
ELECTRON MICROSCOPYf_chiral_restr0.0451527
ELECTRON MICROSCOPYf_plane_restr0.0051627

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