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- PDB-6x0o: Single-Particle Cryo-EM Structure of Arabinosyltransferase EmbB f... -

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Basic information

Entry
Database: PDB / ID: 6x0o
TitleSingle-Particle Cryo-EM Structure of Arabinosyltransferase EmbB from Mycobacterium smegmatis
ComponentsIntegral membrane indolylacetylinositol arabinosyltransferase EmbB
KeywordsMEMBRANE PROTEIN / Glycosyltransferase / nanodisc
Function / homology
Function and homology information


indolylacetylinositol arabinosyltransferase / indolylacetylinositol arabinosyltransferase activity / arabinosyltransferase activity / Actinobacterium-type cell wall biogenesis / Transferases; Glycosyltransferases; Pentosyltransferases / cell wall organization / plasma membrane
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
Similarity search - Domain/homology
1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / Probable arabinosyltransferase A / Integral membrane indolylacetylinositol arabinosyltransferase EmbB
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsTan, Y.Z. / Rodrigues, J. / Keener, J.E. / Zheng, R.B. / Brunton, R. / Kloss, B. / Giacometti, S.I. / Rosario, A.L. / Zhang, L. / Niederweis, M. ...Tan, Y.Z. / Rodrigues, J. / Keener, J.E. / Zheng, R.B. / Brunton, R. / Kloss, B. / Giacometti, S.I. / Rosario, A.L. / Zhang, L. / Niederweis, M. / Clarke, O.B. / Lowary, T.L. / Marty, M.T. / Archer, M. / Potter, C.S. / Carragher, B. / Mancia, F.
Funding support United States, Portugal, European Union, Canada, 12items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM128624 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM111980 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM132120 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R21 AI119672 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103310 United States
Fundacao para a Ciencia e a TecnologiaPD/BD/128261/2016 Portugal
Fundacao para a Ciencia e a TecnologiaPTDC/BIA-BQM/30421/2017 Portugal
Fundacao para a Ciencia e a TecnologiaIF/00656/2014 Portugal
Marie Sklodowska-Curie Actions, FragNET ITNNo 731005European Union
Marie Sklodowska-Curie Actions, FragNET ITNNo 823780European Union
Canadian Glycomics Network (GLYCONET) Canada
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM116799 United States
CitationJournal: Nat Commun / Year: 2020
Title: Cryo-EM structure of arabinosyltransferase EmbB from Mycobacterium smegmatis.
Authors: Yong Zi Tan / José Rodrigues / James E Keener / Ruixiang Blake Zheng / Richard Brunton / Brian Kloss / Sabrina I Giacometti / Ana L Rosário / Lei Zhang / Michael Niederweis / Oliver B ...Authors: Yong Zi Tan / José Rodrigues / James E Keener / Ruixiang Blake Zheng / Richard Brunton / Brian Kloss / Sabrina I Giacometti / Ana L Rosário / Lei Zhang / Michael Niederweis / Oliver B Clarke / Todd L Lowary / Michael T Marty / Margarida Archer / Clinton S Potter / Bridget Carragher / Filippo Mancia /
Abstract: Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti- ...Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single-particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding.
History
DepositionMay 17, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 8, 2020Group: Database references / Structure summary / Category: citation / struct
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _struct.title
Revision 1.2Jul 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Mar 6, 2024Group: Author supporting evidence / Data collection ...Author supporting evidence / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_audit_support / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_audit_support.country

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)120,3615
Polymers118,8351
Non-polymers1,5264
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Monomer
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2550 Å2
ΔGint-36 kcal/mol
Surface area44500 Å2

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Components

#1: Protein Integral membrane indolylacetylinositol arabinosyltransferase EmbB


Mass: 118835.375 Da / Num. of mol.: 1
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: embB, MSMEI_6221 / Production host: Escherichia coli (E. coli)
References: UniProt: I7GAQ2, UniProt: A0R614*PLUS, indolylacetylinositol arabinosyltransferase
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-LHG / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE


Mass: 722.970 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C38H75O10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
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: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.119 MDa / Experimental value: YES
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 278 K

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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 / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording

Imaging-ID: 1 / Detector mode: COUNTING / Num. of grids imaged: 1

IDAverage exposure time (sec.)Electron dose (e/Å2)Film or detector modelNum. of real images
186.478.02FEI FALCON III (4k x 4k)2158
2877.53GATAN K2 SUMMIT (4k x 4k)7833
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 80

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Processing

EM software
IDNameVersionCategoryImage processing-ID
1Gautomatch0.53particle selection1
2Leginon3.4image acquisition
4cryoSPARC2CTF correction1
5RELION2.1CTF correction1
6cisTEM1CTF correction1
9UCSF Chimera1.13model fitting
10Coot0.8model fitting
12cryoSPARC2initial Euler assignment1
13cryoSPARC2final Euler assignment1
15cryoSPARC23D reconstruction1
16Gautomatch0.53particle selection2
18cryoSPARC2CTF correction2
19RELION2.1CTF correction2
20cisTEM1CTF correction2
21cryoSPARC2initial Euler assignment2
22cryoSPARC2final Euler assignment2
24cryoSPARC23D reconstruction2
25PHENIX1.14model refinement
Image processing
IDImage recording-ID
11
22
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
IDImage processing-IDNum. of particles selected
11162271
22700201
Symmetry
IDImage processing-IDEntry-IDPoint symmetry
116X0OC1 (asymmetric)
226X0OC1 (asymmetric)
3D reconstruction

Entry-ID: 6X0O / Num. of particles: 57970 / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Symmetry type: POINT

IDImage processing-IDDetails
11Combined with K2 dataset
22Combined with Falcon III dataset
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingPDB-ID: 3PTY

3pty


Accession code: 3PTY / Source name: PDB / Type: experimental model

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