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- EMDB-30217: Cryo-EM structure of Mycobacterium smegmatis arabinosyltransferas... -

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Basic information

Entry
Database: EMDB / ID: EMD-30217
TitleCryo-EM structure of Mycobacterium smegmatis arabinosyltransferase EmbC2-AcpM2 in complex with ethambutol
Map data
Sample
  • Complex: Mycobacterium smegmatis arabinosyltransferase EmbC2-AcpM2 in complex with ethambutol
    • Complex: EmbC
      • Protein or peptide: Integral membrane indolylacetylinositol arabinosyltransferase EmbC
    • Complex: AcpM
      • Protein or peptide: Meromycolate extension acyl carrier protein
  • Ligand: CALCIUM IONCalcium
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: Ethambutol
  • Ligand: N~3~-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-N-(2-sulfanylethyl)-beta-alaninamide
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
Meromycolate extension acyl carrier protein / Probable arabinosyltransferase A / Integral membrane indolylacetylinositol arabinosyltransferase EmbC
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria) / Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsZhang L / Zhao Y / Gao Y / Wang Q / Li J / Besra GS / Rao Z
Funding support China, United Kingdom, 4 items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB29020000 China
Ministry of Science and Technology (MoST, China)2017YFC0840300 China
National Natural Science Foundation of China (NSFC)81520108019 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, 2020-
Header (metadata) releaseApr 29, 2020-
Map releaseApr 29, 2020-
UpdateJul 1, 2020-
Current statusJul 1, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bve
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30217.png

Downloads & links

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Map

FileDownload / File: emd_30217.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.3
Minimum - Maximum-1.5363176 - 2.9697719
Average (Standard dev.)0.00008829057 (±0.06942023)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 295.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z295.200295.200295.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-1.5362.9700.000

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Supplemental data

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Mask #1

Fileemd_30217_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_30217_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_30217_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Mycobacterium smegmatis arabinosyltransferase EmbC2-AcpM2 in comp...

EntireName: Mycobacterium smegmatis arabinosyltransferase EmbC2-AcpM2 in complex with ethambutol
Components
  • Complex: Mycobacterium smegmatis arabinosyltransferase EmbC2-AcpM2 in complex with ethambutol
    • Complex: EmbC
      • Protein or peptide: Integral membrane indolylacetylinositol arabinosyltransferase EmbC
    • Complex: AcpM
      • Protein or peptide: Meromycolate extension acyl carrier protein
  • Ligand: CALCIUM IONCalcium
  • Ligand: PHOSPHATE IONPhosphate
  • Ligand: Ethambutol
  • Ligand: N~3~-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-N-(2-sulfanylethyl)-beta-alaninamide

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Supramolecule #1: Mycobacterium smegmatis arabinosyltransferase EmbC2-AcpM2 in comp...

SupramoleculeName: Mycobacterium smegmatis arabinosyltransferase EmbC2-AcpM2 in complex with ethambutol
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: EmbC

SupramoleculeName: EmbC / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Supramolecule #3: AcpM

SupramoleculeName: AcpM / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Macromolecule #1: Integral membrane indolylacetylinositol arabinosyltransferase EmbC

MacromoleculeName: Integral membrane indolylacetylinositol arabinosyltransferase EmbC
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: indolylacetylinositol arabinosyltransferase
Source (natural)Organism: Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155
Molecular weightTheoretical: 116.878898 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MTGPHAAGGS NHRTARLVAI IAGLLGTLMA IATPLLPVEQ TTAELNWPQN GVWQSVDAPL IGYVATDLTV TVPCQAAAGL VGPENRNRS VLLSTVPKQA PKAIDRGLLI ERINNDLTVI VRNTPVVSAP LEQVLSPDCR YLTFTAHADK VTGEFVGLTQ G PDDDDPGE ...String:
MTGPHAAGGS NHRTARLVAI IAGLLGTLMA IATPLLPVEQ TTAELNWPQN GVWQSVDAPL IGYVATDLTV TVPCQAAAGL VGPENRNRS VLLSTVPKQA PKAIDRGLLI ERINNDLTVI VRNTPVVSAP LEQVLSPDCR YLTFTAHADK VTGEFVGLTQ G PDDDDPGE AVRGERSGYD FRPQIVGVFT DLSGPAPEGL QLSATIDTRY STSPTLLKLL AMIVGVAMTV IALGALHVLD CA DGRRHKR FLPSRWWSMT PLDGLVSAML VWWHFVGANT ADDGYILTMA RVSEHAGYMA NYYRWFGTPE SPFGWYYDLL ALW AHVSTA SVWMRFPTLL MGLACWWVIS REVIPRLGAA AKHSRAAAWT AAGLFLAFWL PLNNGLRPEP IIALGILLTW CSVE RGVAT SRLLPVAVAI IIGALTLFSG PTGIAAVGAL LVAIGPLKTI VAAHVSRFGY WALLAPIAAA GTVTIFLIFR DQTLA AELQ ASSFKSAVGP SLAWFDEHIR YSRLFTTSPD GSVARRFAVL TLLLALAVSI AMTLRKGRIP GTALGPSRRI IGITII SFL AMMFTPTKWT HHFGVFAGLA GCLGALAAVA VTTTAMKSRR NRTVFGAAVL FVTALSFATV NGWWYVSNFG VPWSNSF PE FKFGFTTMLL GLSVLALLVA AWFHFSGRDV SPDRPQRRWQ RLLVAPLAVA TWALVIFEVV SLTLGMINQY PAWSVGRS N LNALTGKTCG LANDVLVEQN ANAGMLTPIG EPAGQALGAV TSLGFGPNGI PSDVSADPVM EQPGTDNFAD SDSGVVTGT EVGTEGGTTA AAGINGSRAR LPYGLNPATT PVLGSWRSGT QQPAVLRSAW YRLPDRDQAG PLLVVSAAGR FDQGEVEVQW ATDEQAAAN EPGGSITFGD VGAAPAWRNL RAPLSSIPPE ATQIRLVASD DDLAPQHWIA LTPPRIPELR TLQEVVGSSD P VMLDWLVG LAFPCQRPFD HRYGVVEVPK WRILPDRFGA EANSPVMDYL GGGPLGITEL LLRPSSVPTY LKDDWYRDWG SL QRLTPWY PDAQPARLDL GTATRSGWWS PAPLRLSHHH HHHHHHH

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Macromolecule #2: Meromycolate extension acyl carrier protein

MacromoleculeName: Meromycolate extension acyl carrier protein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 10.743876 KDa
SequenceString:
MAATQEEIIA GLAEIIEEVT GIEPSEVTPE KSFVDDLDID SLSMVEIAVQ TEDKYGVKIP DEDLAGLRTV GDVVAYIQKL EEENPEAAA ALREKFAADQ

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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Macromolecule #5: Ethambutol

MacromoleculeName: Ethambutol / type: ligand / ID: 5 / Number of copies: 2 / Formula: 95E
Molecular weightTheoretical: 204.31 Da
Chemical component information

ChemComp-95E:
Ethambutol / medication*YM / Ethambutol

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Macromolecule #6: N~3~-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-N-(2-...

MacromoleculeName: N~3~-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-N-(2-sulfanylethyl)-beta-alaninamide
type: ligand / ID: 6 / Number of copies: 2 / Formula: PN7
Molecular weightTheoretical: 358.348 Da
Chemical component information

ChemComp-PN7:
N~3~-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-N-(2-sulfanylethyl)-beta-alaninamide

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormula
20.0 mMHepes
150.0 mMNaClSodium chloride
0.04 w/vGDN
VitrificationCryogen name: ETHANE
DetailsThe sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 78.5 K / Max: 78.6 K
Alignment procedureComa free - Residual tilt: 10.0 mrad
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 5100 / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1855947
CTF correctionSoftware - Name: cryoSPARC (ver. 2.9.1)
Details: The CTF correction was done by patch CTF correction.
Startup modelType of model: NONE / Details: The startup models were generated automatically.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9.1)
Details: The initial angle assignment was generated by Ab-initio reconstruction.
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 2.9.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9.1)
Details: The final angle assignment was non-uniform refinement.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 217550
DetailsThe selected images were normalized.

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT / Overall B value: 62.01 / Target criteria: correlation coefficient
Output model

PDB-7bve:
Cryo-EM structure of Mycobacterium smegmatis arabinosyltransferase EmbC2-AcpM2 in complex with ethambutol

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