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- EMDB-35410: Arabinosyltransferase AftA -

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Basic information

Entry
Database: EMDB / ID: EMD-35410
TitleArabinosyltransferase AftA
Map dataarabinosyltransferase dimer, C1 symmetry
Sample
  • Complex: AftA dimer
    • Protein or peptide: Galactan 5-O-arabinofuranosyltransferase
  • Ligand: CALCIUM IONCalcium
KeywordsArabinosyltransferase / AftA / Mycobacterium tuberculosis / MEMBRANE PROTEIN
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 H37Rv (bacteria) / Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsGong YC / Rao ZH / Zhang L
Funding support China, 1 items
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, 2023-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_35410.png

Downloads & links

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Map

FileDownload / File: emd_35410.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationarabinosyltransferase dimer, C1 symmetry
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.43
Minimum - Maximum-2.3154504 - 3.2815635
Average (Standard dev.)0.0012406944 (±0.06147498)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 319.488 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: arabinosyltransferase dimer, C1 symmetry, half mapA

Fileemd_35410_half_map_1.map
Annotationarabinosyltransferase dimer, C1 symmetry, half mapA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: arabinosyltransferase dimer, C1 symmetry, half mapB

Fileemd_35410_half_map_2.map
Annotationarabinosyltransferase dimer, C1 symmetry, half mapB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AftA dimer

EntireName: AftA dimer
Components
  • Complex: AftA dimer
    • Protein or peptide: Galactan 5-O-arabinofuranosyltransferase
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: AftA dimer

SupramoleculeName: AftA dimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: AftA subunit1, AftA subunit2
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 69.5 KDa

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Macromolecule #1: Galactan 5-O-arabinofuranosyltransferase

MacromoleculeName: Galactan 5-O-arabinofuranosyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: galactan 5-O-arabinofuranosyltransferase
Source (natural)Organism: Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Molecular weightTheoretical: 69.576938 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MPSRRKSPQF GHEMGAFTSA RAREVLVALG QLAAAVVVAV GVAVVSLLAI ARVEWPAFPS SNQLHALTTV GQVGCLAGLV GIGWLWRHG RFRRLARLGG LVLVSAFTVV TLGMPLGATK LYLFGISVDQ QFRTEYLTRL TDTAALRDMT YIGLPPFYPP G WFWIGGRA ...String:
MPSRRKSPQF GHEMGAFTSA RAREVLVALG QLAAAVVVAV GVAVVSLLAI ARVEWPAFPS SNQLHALTTV GQVGCLAGLV GIGWLWRHG RFRRLARLGG LVLVSAFTVV TLGMPLGATK LYLFGISVDQ QFRTEYLTRL TDTAALRDMT YIGLPPFYPP G WFWIGGRA AALTGTPAWE MFKPWAITSM AIAVAVALVL WWRMIRFEYA LLVTVATAAV MLAYSSPEPY AAMITVLLPP ML VLTWSGL GARDRQGWAA VVGAGVFLGF AATWYTLLVA YGAFTVVLMA LLLAGSRLQS GIKAAVDPLC RLAVVGAIAA AIG STTWLP YLLRAARDPV SDTGSAQHYL PADGAALTFP MLQFSLLGAI CLLGTLWLVM RARSSAPAGA LAIGVLAVYL WSLL SMLAT LARTTLLSFR LQPTLSVLLV AAGAFGFVEA VQALGKRGRG VIPMAAAIGL AGAIAFSQDI PDVLRPDLTI AYTDT DGYG QRGDRRPPGS EKYYPAIDAA IRRVTGKRRD RTVVLTADYS FLSYYPYWGF QGLTPHYANP LAQFDKRATQ IDSWSG LST ADEFIAALDK LPWQPPTVFL MRHGAHNSYT LRLAQDVYPN QPNVRRYTVD LRTALFADPR FVVEDIGPFV LAIRKPQ ES A

UniProtKB: Galactan 5-O-arabinofuranosyltransferase

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration6.5 mg/mL
BufferpH: 8 / Component - Concentration: 150.0 mM / Component - Formula: NaClSodium chloride / Component - Name: sodium chloride / Details: 150mM NaCl, 20mM Hepes, 0.04%GDN
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Detailsthe sample was mono disperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 59000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 10500
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 6113 / Number real images: 6113 / Average exposure time: 2.4 sec. / Average electron dose: 60.0 e/Å2 / Details: images were collected in high resolution mode
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2752481
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 147896

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