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- PDB-7bvf: Cryo-EM structure of Mycobacterium tuberculosis arabinosyltransfe... -

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Basic information

Entry
Database: PDB / ID: 7bvf
TitleCryo-EM structure of Mycobacterium tuberculosis arabinosyltransferase EmbA-EmbB-AcpM2 in complex with ethambutol
Components
  • (Probable arabinosyltransferase ...) x 2
  • Meromycolate extension acyl carrier protein
KeywordsTRANSFERASE / Mycobacterium tuberculosis / cell wall synthesis / drug target / ethambutol / arabinosyltransferase / EmbA / EmbB / EmbC / acyl carrier protein / arabinogalactan / lipoarabinomannan / drug resistance
Function / homology
Function and homology information


arabinosyltransferase activity / Actinobacterium-type cell wall biogenesis / cell wall / plasma membrane => GO:0005886 / acyl carrier activity / Transferases; Glycosyltransferases; Pentosyltransferases / cell wall organization / membrane => GO:0016020 / response to antibiotic / plasma membrane ...arabinosyltransferase activity / Actinobacterium-type cell wall biogenesis / cell wall / plasma membrane => GO:0005886 / acyl carrier activity / Transferases; Glycosyltransferases; Pentosyltransferases / cell wall organization / membrane => GO:0016020 / response to antibiotic / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Arabinofuranosyltransferase, central domain / Arabinofuranosyltransferase, domain 1 / Arabinosyltransferase, C-terminal / Arabinosyltransferas, concanavalin like domain / Arabinosyltransferase, C-terminal, subdomain 2 / Mycobacterial cell wall arabinan synthesis protein / EmbC C-terminal domain / Arabinosyltransferase concanavalin like domain / Acyl carrier protein (ACP) / Phosphopantetheine attachment site ...Arabinofuranosyltransferase, central domain / Arabinofuranosyltransferase, domain 1 / Arabinosyltransferase, C-terminal / Arabinosyltransferas, concanavalin like domain / Arabinosyltransferase, C-terminal, subdomain 2 / Mycobacterial cell wall arabinan synthesis protein / EmbC C-terminal domain / Arabinosyltransferase concanavalin like domain / Acyl carrier protein (ACP) / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain
Similarity search - Domain/homology
Ethambutol / CARDIOLIPIN / MONO-TRANS, OCTA-CIS DECAPRENYL-PHOSPHATE / Meromycolate extension acyl carrier protein / Probable arabinosyltransferase B / Probable arabinosyltransferase A
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
Mycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsZhang, L. / Zhao, Y. / Gao, Y. / Wang, Q. / Li, J. / Besra, G.S. / Rao, Z.
Funding support China, United Kingdom, 4items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB29020000 China
National Natural Science Foundation of China (NSFC)81520108019 China
Ministry of Science and Technology (MoST, China)2017YFC0840300 China
Medical Research Council (MRC, United Kingdom)MR/S000542/1 United Kingdom
CitationJournal: Science / Year: 2020
Title: Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol.
Authors: Lu Zhang / Yao Zhao / Yan Gao / Lijie Wu / Ruogu Gao / Qi Zhang / Yinan Wang / Chengyao Wu / Fangyu Wu / Sudagar S Gurcha / Natacha Veerapen / Sarah M Batt / Wei Zhao / Ling Qin / Xiuna Yang ...Authors: Lu Zhang / Yao Zhao / Yan Gao / Lijie Wu / Ruogu Gao / Qi Zhang / Yinan Wang / Chengyao Wu / Fangyu Wu / Sudagar S Gurcha / Natacha Veerapen / Sarah M Batt / Wei Zhao / Ling Qin / Xiuna Yang / Manfu Wang / Yan Zhu / Bing Zhang / Lijun Bi / Xian'en Zhang / Haitao Yang / Luke W Guddat / Wenqing Xu / Quan Wang / Jun Li / Gurdyal S Besra / Zihe Rao /
Abstract: The arabinosyltransferases EmbA, EmbB, and EmbC are involved in cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo- ...The arabinosyltransferases EmbA, EmbB, and EmbC are involved in cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo-electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.
History
DepositionApr 10, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 29, 2020Provider: repository / Type: Initial release
Revision 1.1May 13, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 1, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
B: Probable arabinosyltransferase B
A: Probable arabinosyltransferase A
P: Meromycolate extension acyl carrier protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)251,8178
Polymers247,8663
Non-polymers3,9525
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
DetailsThe complete assembly should be tetramer. There should be 2 molecules of AcpM, however, only one was modeled due to low density.

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Components

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Probable arabinosyltransferase ... , 2 types, 2 molecules BA

#1: Protein Probable arabinosyltransferase B


Mass: 120332.398 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: embB, Rv3795, MTCY13D12.29
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: P9WNL7, Transferases; Glycosyltransferases; Pentosyltransferases
#2: Protein Probable arabinosyltransferase A


Mass: 116789.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: embA, Rv3794, MTCY13D12.28
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: P9WNL9, Transferases; Glycosyltransferases; Pentosyltransferases

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Protein , 1 types, 1 molecules P

#3: Protein Meromycolate extension acyl carrier protein / ACP


Mass: 10743.876 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0R0B3

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Non-polymers , 4 types, 5 molecules

#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-95E / Ethambutol / Ethambutol


Mass: 204.310 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H24N2O2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#6: Chemical ChemComp-DSL / MONO-TRANS, OCTA-CIS DECAPRENYL-PHOSPHATE


Mass: 779.165 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C50H83O4P / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-CDL / CARDIOLIPIN / DIPHOSPHATIDYL GLYCEROL / BIS-(1,2-DIACYL-SN-GLYCERO-3-PHOSPHO)-1',3'-SN-GLYCEROL / Cardiolipin


Mass: 1464.043 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C81H156O17P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1Mycobacterium tuberculosis arabinosyltransferase EmbA-EmbB-AcpM2 in complex with ethambutolCOMPLEX#1-#30MULTIPLE SOURCES
2EmbA and EmbBCOMPLEX#1-#31RECOMBINANT
3AcpMCOMPLEX#31NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mycobacterium tuberculosis H37Rv (bacteria)83332
23Mycolicibacterium smegmatis MC2 155 (bacteria)246196
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Buffer solutionpH: 7.4
Buffer component
IDConc.FormulaBuffer-ID
120 mMHepes1
2150 mMNaClSodium chloride1
30.04 w/vGDN1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse.
Specimen supportGrid material: COPPER / Grid type: Quantifoil R0.6/1
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 78.6 K / Temperature (min): 78.5 K / Residual tilt: 10 mradians
Image recording
IDImaging-IDAverage exposure time (sec.)Electron dose (e/Å2)Film or detector modelNum. of grids imagedNum. of real images
11250GATAN K3 (6k x 4k)17612
2150GATAN K3 (6k x 4k)
3150GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEM3-7-3image acquisition
7UCSF Chimera1.14model fitting
13PHENIX1.16model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 521803 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 32.28 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005317371
ELECTRON MICROSCOPYf_angle_d0.777923765
ELECTRON MICROSCOPYf_chiral_restr0.04842751
ELECTRON MICROSCOPYf_plane_restr0.00613018
ELECTRON MICROSCOPYf_dihedral_angle_d17.068810248

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