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- PDB-7bvg: Cryo-EM structure of Mycobacterium smegmatis arabinosyltransferas... -

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Basic information

Entry
Database: PDB / ID: 7bvg
TitleCryo-EM structure of Mycobacterium smegmatis arabinosyltransferase EmbA-EmbB-AcpM2 in complex with di-arabinose.
Components
  • (Integral membrane indolylacetylinositol arabinosyltransferase ...) x 2
  • Meromycolate extension acyl carrier protein
KeywordsTRANSFERASE / Mycobacterium smegmatis / cell wall synthesis / drug target / ethambutol / arabinosyltransferase / EmbA / EmbB / EmbC / acyl carrier protein / arabinogalactan / lipoarabinomannan / drug resistance
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
CARDIOLIPIN / Chem-F8L / 4'-PHOSPHOPANTETHEINE / PHOSPHATE ION / Meromycolate extension acyl carrier protein / Probable arabinosyltransferase B / 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: 3.1 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 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / entity ...atom_site / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / pdbx_unobs_or_zero_occ_atoms / struct_asym / struct_conn / struct_conn_type / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.src_method / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Mar 27, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2
Item: _chem_comp.pdbx_synonyms / _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 EmbA
B: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
P: Meromycolate extension acyl carrier protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)251,03911
Polymers246,3833
Non-polymers4,6558
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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Integral membrane indolylacetylinositol arabinosyltransferase ... , 2 types, 2 molecules AB

#1: Protein Integral membrane indolylacetylinositol arabinosyltransferase EmbA / Probable arabinosyltransferase B


Mass: 116561.898 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: embA
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0R613, Transferases; Glycosyltransferases; Pentosyltransferases
#2: Protein Integral membrane indolylacetylinositol arabinosyltransferase EmbB


Mass: 119077.672 Da / Num. of mol.: 1
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

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Protein / Sugars , 2 types, 2 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
#4: Polysaccharide alpha-D-arabinofuranose-(1-5)-alpha-D-arabinofuranose


Type: oligosaccharide / Mass: 282.245 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DArafa1-5DArafa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a122h-1a_1-4]/1-1/a5-b1WURCSPDB2Glycan 1.1.0
[][a-D-Araf]{[(5+1)][a-D-Araf]{}}LINUCSPDB-CARE

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Non-polymers , 5 types, 7 molecules

#5: 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
#6: 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
#7: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: Ca
#8: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#9: Chemical ChemComp-PNS / 4'-PHOSPHOPANTETHEINE / Phosphopantetheine


Mass: 358.348 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H23N2O7PS / Feature type: SUBJECT OF INVESTIGATION

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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 smegmatis arabinosyltransferase EmbA-EmbB-AcpM2 in complex with di-arabinoseCOMPLEX#1-#30MULTIPLE SOURCES
2EmbA and EmbBCOMPLEX#1-#21RECOMBINANT
3AcpMCOMPLEX#31NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mycolicibacterium smegmatis MC2 155 (bacteria)246196
23Mycolicibacterium smegmatis MC2 155 (bacteria)246196
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Buffer solutionpH: 7.4
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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
Image recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 209894 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 56.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003617660
ELECTRON MICROSCOPYf_angle_d0.708624149
ELECTRON MICROSCOPYf_chiral_restr0.04582769
ELECTRON MICROSCOPYf_plane_restr0.00543086
ELECTRON MICROSCOPYf_dihedral_angle_d17.305210439

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