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

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Basic information

Entry
Database: PDB / ID: 7bve
TitleCryo-EM structure of Mycobacterium smegmatis arabinosyltransferase EmbC2-AcpM2 in complex with ethambutol
Components
  • Integral membrane indolylacetylinositol arabinosyltransferase EmbC
  • Meromycolate extension acyl carrier protein
KeywordsTRANSFERASE / Mycobacterium smegmatis / cell wall synthesis / drug target / ethambutol / arabinosyltransferase / 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 / lipid A biosynthetic process / acyl binding / acyl carrier activity / Transferases; Glycosyltransferases; Pentosyltransferases / cell wall organization / metal ion binding ...indolylacetylinositol arabinosyltransferase / indolylacetylinositol arabinosyltransferase activity / arabinosyltransferase activity / Actinobacterium-type cell wall biogenesis / lipid A biosynthetic process / acyl binding / acyl carrier activity / Transferases; Glycosyltransferases; Pentosyltransferases / cell wall organization / metal ion binding / plasma membrane / cytosol
Similarity search - Function
arabinofuranosyltransferase like domain / : / 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 ...arabinofuranosyltransferase like domain / : / 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 / Jelly Rolls / Sandwich / Mainly Beta
Similarity search - Domain/homology
Ethambutol / Chem-PN7 / PHOSPHATE ION / Meromycolate extension acyl carrier protein / Probable arabinosyltransferase A / Integral membrane indolylacetylinositol arabinosyltransferase EmbC
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (bacteria)
Mycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.81 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 1.3Nov 6, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / struct_conn / struct_conn_type
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification / _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

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-30217
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)256,64112
Polymers255,2464
Non-polymers1,3958
Water00
1


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

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Components

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Protein , 2 types, 4 molecules ABCD

#1: Protein Integral membrane indolylacetylinositol arabinosyltransferase EmbC


Mass: 116878.898 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155 / Gene: embC, MSMEI_6219
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: I7FMU5, UniProt: A0R612*PLUS, indolylacetylinositol arabinosyltransferase
#2: Protein Meromycolate extension acyl carrier protein / ACP


Mass: 10743.876 Da / Num. of mol.: 2 / 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, 8 molecules

#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#5: Chemical ChemComp-95E / Ethambutol


Mass: 204.310 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H24N2O2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#6: Chemical ChemComp-PN7 / N~3~-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-N-(2-sulfanylethyl)-beta-alaninamide


Mass: 358.348 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C11H23N2O7PS

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Details

Has ligand of interestY
Has protein modificationY

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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 EmbC2-AcpM2 in complex with ethambutolCOMPLEX#1-#20MULTIPLE SOURCES
2EmbCCOMPLEX#11RECOMBINANT
3AcpMCOMPLEX#21NATURAL
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
Buffer component
IDConc.FormulaBuffer-ID
120 mMHepes1
2150 mMNaCl1
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.
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 FIELD / Nominal magnification: 29000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2000 nm / 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 recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5100

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Processing

EM software
IDNameVersionCategory
2SerialEM3-7-3image acquisition
4cryoSPARC2.9.1CTF correction
7UCSF Chimera1.14model fitting
9PHENIX1.16model refinement
10cryoSPARC2.9.1initial Euler assignment
11cryoSPARC2.9.1final Euler assignment
12cryoSPARC2.9.1classification
13cryoSPARC3D reconstruction
Image processingDetails: The selected images were normalized.
CTF correctionDetails: The CTF correction was done by patch CTF correction.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1855947
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 217550 / Symmetry type: POINT
Atomic model buildingB value: 62.01 / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient

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