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- EMDB-30218: Cryo-EM structure of Mycobacterium tuberculosis arabinosyltransfe... -

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Basic information

Entry
Database: EMDB / ID: EMD-30218
TitleCryo-EM structure of Mycobacterium tuberculosis arabinosyltransferase EmbA-EmbB-AcpM2 in complex with ethambutol
Map data
Sample
  • Complex: Mycobacterium tuberculosis arabinosyltransferase EmbA-EmbB-AcpM2 in complex with ethambutol
    • Complex: EmbA and EmbB
      • Protein or peptide: Probable arabinosyltransferase B
      • Protein or peptide: Probable arabinosyltransferase A
      • Protein or peptide: Meromycolate extension acyl carrier protein
    • Complex: AcpM
  • Ligand: CALCIUM ION
  • Ligand: Ethambutol
  • Ligand: MONO-TRANS, OCTA-CIS DECAPRENYL-PHOSPHATE
  • Ligand: CARDIOLIPIN
KeywordsMycobacterium tuberculosis / cell wall synthesis / drug target / ethambutol / arabinosyltransferase / EmbA / EmbB / EmbC / acyl carrier protein / arabinogalactan / lipoarabinomannan / drug resistance / TRANSFERASE
Function / homology
Function and homology information


arabinosyltransferase activity / Actinobacterium-type cell wall biogenesis / lipid A biosynthetic process / acyl binding / acyl carrier activity / Transferases; Glycosyltransferases; Pentosyltransferases / peptidoglycan-based cell wall / cell wall organization / response to antibiotic / membrane ...arabinosyltransferase activity / Actinobacterium-type cell wall biogenesis / lipid A biosynthetic process / acyl binding / acyl carrier activity / Transferases; Glycosyltransferases; Pentosyltransferases / peptidoglycan-based cell wall / cell wall organization / response to antibiotic / membrane / plasma membrane / cytosol
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) ...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 B / Probable arabinosyltransferase A
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria) / Mycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsZhang L / Zhao Y
Funding support China, United Kingdom, 4 items
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, 2020-
Header (metadata) releaseApr 29, 2020-
Map releaseApr 29, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.28
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.28
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7bvf
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30218.png

Downloads & links

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Map

FileDownload / File: emd_30218.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 384 pix.
= 314.88 Å		0.82 Å/pix.
x 384 pix.
= 314.88 Å		0.82 Å/pix.
x 384 pix.
= 314.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.28
Minimum - Maximum-1.8078153 - 3.0105896
Average (Standard dev.)-0.0005309899 (±0.069157675)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 314.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z314.880314.880314.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-1.8083.011-0.001

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Supplemental data

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Mask #1

Fileemd_30218_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_30218_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_30218_half_map_2.map
Projections & Slices
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Sample components

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Entire : Mycobacterium tuberculosis arabinosyltransferase EmbA-EmbB-AcpM2 ...

EntireName: Mycobacterium tuberculosis arabinosyltransferase EmbA-EmbB-AcpM2 in complex with ethambutol
Components
  • Complex: Mycobacterium tuberculosis arabinosyltransferase EmbA-EmbB-AcpM2 in complex with ethambutol
    • Complex: EmbA and EmbB
      • Protein or peptide: Probable arabinosyltransferase B
      • Protein or peptide: Probable arabinosyltransferase A
      • Protein or peptide: Meromycolate extension acyl carrier protein
    • Complex: AcpM
  • Ligand: CALCIUM ION
  • Ligand: Ethambutol
  • Ligand: MONO-TRANS, OCTA-CIS DECAPRENYL-PHOSPHATE
  • Ligand: CARDIOLIPIN

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Supramolecule #1: Mycobacterium tuberculosis arabinosyltransferase EmbA-EmbB-AcpM2 ...

SupramoleculeName: Mycobacterium tuberculosis arabinosyltransferase EmbA-EmbB-AcpM2 in complex with ethambutol
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: EmbA and EmbB

SupramoleculeName: EmbA and EmbB / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)

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Supramolecule #3: AcpM

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

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Macromolecule #1: Probable arabinosyltransferase B

MacromoleculeName: Probable arabinosyltransferase B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Glycosyltransferases; Pentosyltransferases
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 120.332398 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MTQCASRRKS TPNRAILGAF ASARGTRWVA TIAGLIGFVL SVATPLLPVV QTTAMLDWPQ RGQLGSVTAP LISLTPVDFT ATVPCDVVR AMPPAGGVVL GTAPKQGKDA NLQALFVVVS AQRVDVTDRN VVILSVPREQ VTSPQCQRIE VTSTHAGTFA N FVGLKDPS ...String:
MTQCASRRKS TPNRAILGAF ASARGTRWVA TIAGLIGFVL SVATPLLPVV QTTAMLDWPQ RGQLGSVTAP LISLTPVDFT ATVPCDVVR AMPPAGGVVL GTAPKQGKDA NLQALFVVVS AQRVDVTDRN VVILSVPREQ VTSPQCQRIE VTSTHAGTFA N FVGLKDPS GAPLRSGFPD PNLRPQIVGV FTDLTGPAPP GLAVSATIDT RFSTRPTTLK LLAIIGAIVA TVVALIALWR LD QLDGRGS IAQLLLRPFR PASSPGGMRR LIPASWRTFT LTDAVVIFGF LLWHVIGANS SDDGYILGMA RVADHAGYMS NYF RWFGSP EDPFGWYYNL LALMTHVSDA SLWMRLPDLA AGLVCWLLLS REVLPRLGPA VEASKPAYWA AAMVLLTAWM PFNN GLRPE GIIALGSLVT YVLIERSMRY SRLTPAALAV VTAAFTLGVQ PTGLIAVAAL VAGGRPMLRI LVRRHRLVGT LPLVS PMLA AGTVILTVVF ADQTLSTVLE ATRVRAKIGP SQAWYTENLR YYYLILPTVD GSLSRRFGFL ITALCLFTAV FIMLRR KRI PSVARGPAWR LMGVIFGTMF FLMFTPTKWV HHFGLFAAVG AAMAALTTVL VSPSVLRWSR NRMAFLAALF FLLALCW AT TNGWWYVSSY GVPFNSAMPK IDGITVSTIF FALFAIAAGY AAWLHFAPRG AGEGRLIRAL TTAPVPIVAG FMAAVFVA S MVAGIVRQYP TYSNGWSNVR AFVGGCGLAD DVLVEPDTNA GFMKPLDGDS GSWGPLGPLG GVNPVGFTPN GVPEHTVAE AIVMKPNQPG TDYDWDAPTK LTSPGINGST VPLPYGLDPA RVPLAGTYTT GAQQQSTLVS AWYLLPKPDD GHPLVVVTAA GKIAGNSVL HGYTPGQTVV LEYAMPGPGA LVPAGRMVPD DLYGEQPKAW RNLRFARAKM PADAVAVRVV AEDLSLTPED W IAVTPPRV PDLRSLQEYV GSTQPVLLDW AVGLAFPCQQ PMLHANGIAE IPKFRITPDY SAKKLDTDTW EDGTNGGLLG IT DLLLRAH VMATYLSRDW ARDWGSLRKF DTLVDAPPAQ LELGTATRSG LWSPGKIRIG PHLGGIKAFH HHHHHHHHH

UniProtKB: Probable arabinosyltransferase B

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Macromolecule #2: Probable arabinosyltransferase A

MacromoleculeName: Probable arabinosyltransferase A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Glycosyltransferases; Pentosyltransferases
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 116.7895 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: DYKDDDDKVP HDGNERSHRI ARLAAVVSGI AGLLLCGIVP LLPVNQTTAT IFWPQGSTAD GNITQITAPL VSGAPRALDI SIPCSAIAT LPANGGLVLS TLPAGGVDTG KAGLFVRANQ DTVVVAFRDS VAAVAARSTI AAGGCSALHI WADTGGAGAD F MGIPGGAG ...String:
DYKDDDDKVP HDGNERSHRI ARLAAVVSGI AGLLLCGIVP LLPVNQTTAT IFWPQGSTAD GNITQITAPL VSGAPRALDI SIPCSAIAT LPANGGLVLS TLPAGGVDTG KAGLFVRANQ DTVVVAFRDS VAAVAARSTI AAGGCSALHI WADTGGAGAD F MGIPGGAG TLPPEKKPQV GGIFTDLKVG AQPGLSARVD IDTRFITTPG ALKKAVMLLG VLAVLVAMVG LAALDRLSRG RT LRDWLTR YRPRVRVGFA SRLADAAVIA TLLLWHVIGA TSSDDGYLLT VARVAPKAGY VANYYRYFGT TEAPFDWYTS VLA QLAAVS TAGVWMRLPA TLAGIACWLI VSRFVLRRLG PGPGGLASNR VAVFTAGAVF LSAWLPFNNG LRPEPLIALG VLVT WVLVE RSIALGRLAP AAVAIIVATL TATLAPQGLI ALAPLLTGAR AIAQRIRRRR ATDGLLAPLA VLAAALSLIT VVVFR DQTL ATVAESARIK YKVGPTIAWY QDFLRYYFLT VESNVEGSMS RRFAVLVLLF CLFGVLFVLL RRGRVAGLAS GPAWRL IGT TAVGLLLLTF TPTKWAVQFG AFAGLAGVLG AVTAFTFARI GLHSRRNLTL YVTALLFVLA WATSGINGWF YVGNYGV PW YDIQPVIASH PVTSMFLTLS ILTGLLAAWY HFRMDYAGHT EVKDNRRNRI LASTPLLVVA VIMVAGEVGS MAKAAVFR Y PLYTTAKANL TALSTGLSSC AMADDVLAEP DPNAGMLQPV PGQAFGPDGP LGGISPVGFK PEGVGEDLKS DPVVSKPGL VNSDASPNKP NAAITDSAGT AGGKGPVGIN GSHAALPFGL DPARTPVMGS YGENNLAATA TSAWYQLPPR SPDRPLVVVS AAGAIWSYK EDGDFIYGQS LKLQWGVTGP DGRIQPLGQV FPIDIGPQPA WRNLRFPLAW APPEADVARI VAYDPNLSPE Q WFAFTPPR VPVLESLQRL IGSATPVLMD IATAANFPCQ RPFSEHLGIA ELPQYRILPD HKQTAASSNL WQSSSTGGPF LF TQALLRT STIATYLRGD WYRDWGSVEQ YHRLVPADQA PDAVVEEGVI TVPGWGRPGP IRALP

UniProtKB: Probable arabinosyltransferase A

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Macromolecule #3: Meromycolate extension acyl carrier protein

MacromoleculeName: Meromycolate extension acyl carrier protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / 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

UniProtKB: Meromycolate extension acyl carrier protein

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: Ethambutol

MacromoleculeName: Ethambutol / type: ligand / ID: 5 / Number of copies: 1 / Formula: 95E
Molecular weightTheoretical: 204.31 Da
Chemical component information

ChemComp-95E:
Ethambutol

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Macromolecule #6: MONO-TRANS, OCTA-CIS DECAPRENYL-PHOSPHATE

MacromoleculeName: MONO-TRANS, OCTA-CIS DECAPRENYL-PHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: DSL
Molecular weightTheoretical: 779.165 Da
Chemical component information

ChemComp-DSL:
MONO-TRANS, OCTA-CIS DECAPRENYL-PHOSPHATE

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Macromolecule #7: CARDIOLIPIN

MacromoleculeName: CARDIOLIPIN / type: ligand / ID: 7 / Number of copies: 2 / Formula: CDL
Molecular weightTheoretical: 1.464043 KDa
Chemical component information

ChemComp-CDL:
CARDIOLIPIN / phospholipid*YM

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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 mMNaCl
0.04 w/vGDN
GridModel: Quantifoil R0.6/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE
DetailsThe sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 78.5 K / Max: 78.6 K
Alignment procedureComa free - Residual tilt: 10.0 mrad
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 7612 / #0 - Average exposure time: 2.0 sec. / #0 - Average electron dose: 50.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 (6k x 4k) / #1 - Average electron dose: 50.0 e/Å2 / #2 - Image recording ID: 3 / #2 - Film or detector model: GATAN K3 (6k x 4k) / #2 - Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Image recording ID1
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 521803
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7bvf:
Cryo-EM structure of Mycobacterium tuberculosis arabinosyltransferase EmbA-EmbB-AcpM2 in complex with ethambutol

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