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- PDB-7bwr: Mycobacterium smegmatis arabinosyltransferase complex EmbB2-AcpM2... -

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Basic information

Entry
Database: PDB / ID: 7bwr
TitleMycobacterium smegmatis arabinosyltransferase complex EmbB2-AcpM2 in substrate DPA bound asymmetric "active state"
Components
  • Integral membrane indolylacetylinositol arabinosyltransferase EmbB
  • Meromycolate extension acyl carrier protein
KeywordsTRANSFERASE / Mycobacterium tuberculosis / EmbB / cryo-EM / ethambutol / cell wall synthesis / arabinoglacatan / arabinosyltransferase / acyl-carrier-protein
Function / homology
Function and homology information


indolylacetylinositol arabinosyltransferase / indolylacetylinositol arabinosyltransferase activity / arabinosyltransferase activity / Actinobacterium-type cell wall biogenesis / acyl carrier activity / Transferases; Glycosyltransferases; Pentosyltransferases / cell wall organization / plasma membrane / 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
Chem-F8L / Meromycolate extension acyl carrier protein / Probable arabinosyltransferase A / Integral membrane indolylacetylinositol arabinosyltransferase EmbB
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsGao, R.G. / Zhang, L. / Wang, Q. / Rao, Z.H.
Funding support China, 2items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2017YFC0840300 China
National Natural Science Foundation of China (NSFC)81520108019 China
CitationJournal: Protein Cell / Year: 2020
Title: Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM.
Authors: Lu Zhang / Yao Zhao / Ruogu Gao / Jun Li / Xiuna Yang / Yan Gao / Wei Zhao / Sudagar S Gurcha / Natacha Veerapen / Sarah M Batt / Kajelle Kaur Besra / Wenqing Xu / Lijun Bi / Xian'en Zhang / ...Authors: Lu Zhang / Yao Zhao / Ruogu Gao / Jun Li / Xiuna Yang / Yan Gao / Wei Zhao / Sudagar S Gurcha / Natacha Veerapen / Sarah M Batt / Kajelle Kaur Besra / Wenqing Xu / Lijun Bi / Xian'en Zhang / Luke W Guddat / Haitao Yang / Quan Wang / Gurdyal S Besra / Zihe Rao /
Abstract: Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the ...Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.
History
DepositionApr 15, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 27, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 8, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Mar 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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Assembly

Deposited unit
A: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
B: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
C: Meromycolate extension acyl carrier protein
D: Meromycolate extension acyl carrier protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)256,1806
Polymers255,2284
Non-polymers9512
Water0
1


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

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Components

#1: Protein Integral membrane indolylacetylinositol arabinosyltransferase EmbB


Mass: 116870.289 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: embB, MSMEI_6221
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: I7GAQ2, UniProt: A0R614*PLUS, indolylacetylinositol arabinosyltransferase
#2: Protein Meromycolate extension acyl carrier protein / ACP


Mass: 10743.876 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: acpM, MSMEG_4326, MSMEI_4226
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0R0B3
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-F8L / [(2Z,6E,10E,14Z,18E,22Z,26Z)-3,7,11,15,19,23,27,31,35,39-decamethyltetraconta-2,6,10,14,18,22,26,30,34,38-decaenyl] [(2S,3S,4S,5R)-5-(hydroxymethyl)-3,4-bis(oxidanyl)oxolan-2-yl] hydrogen phosphate


Mass: 911.280 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C55H91O8P / 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: Mycobacterial Arabinosyltransferase Complex EmbB2-AcpM2
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 254 kDa/nm / Experimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Classification: refinement
EM software
IDNameCategory
1Gautomatchparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10REFMACmodel refinement
11RELIONinitial Euler assignment
12cryoSPARCfinal Euler assignment
14cryoSPARC3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 125899 / Symmetry type: POINT
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 178.88 Å2 / Biso mean: 94.9808 Å2 / Biso min: 20 Å2

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