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- EMDB-30234: Mycobacterium smegmatis arabinosyltransferase complex EmbB2-AcpM2... -

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Basic information

Entry
Database: EMDB / ID: EMD-30234
TitleMycobacterium smegmatis arabinosyltransferase complex EmbB2-AcpM2 in substrate DPA bound asymmetric "active state
Map datahalf map B from cryoSPARC
Sample
  • Complex: Mycobacterial Arabinosyltransferase Complex EmbB2-AcpM2
    • Protein or peptide: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
    • Protein or peptide: Meromycolate extension acyl carrier protein
  • Ligand: CALCIUM ION
  • Ligand: [(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
KeywordsMycobacterium tuberculosis / EmbB / cryo-EM / ethambutol / cell wall synthesis / arabinoglacatan / arabinosyltransferase / acyl-carrier-protein / TRANSFERASE
Function / homology
Function and homology information


indolylacetylinositol arabinosyltransferase / indolylacetylinositol arabinosyltransferase activity / arabinosyltransferase activity / Actinobacterium-type cell wall biogenesis / Transferases; Glycosyltransferases; Pentosyltransferases / acyl carrier activity / 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) ...: / 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 EmbB
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsGao RG / Zhang L
Funding support China, 2 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2017YFC0840300 China
National Natural Science Foundation of China (NSFC)81520108019 China
CitationJournal: Protein Cell / Year: 2020
Title: Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM.
Authors: Lu Zhang / Yao Zhao / Ruogu Gao / Jun Li / Xiuna Yang / Yan Gao / Wei Zhao / Sudagar S Gurcha / Natacha Veerapen / Sarah M Batt / Kajelle Kaur Besra / Wenqing Xu / Lijun Bi / Xian'en Zhang / ...Authors: Lu Zhang / Yao Zhao / Ruogu Gao / Jun Li / Xiuna Yang / Yan Gao / Wei Zhao / Sudagar S Gurcha / Natacha Veerapen / Sarah M Batt / Kajelle Kaur Besra / Wenqing Xu / Lijun Bi / Xian'en Zhang / Luke W Guddat / Haitao Yang / Quan Wang / Gurdyal S Besra / Zihe Rao /
Abstract: Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the ...Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.
History
DepositionApr 15, 2020-
Header (metadata) releaseMay 27, 2020-
Map releaseMay 27, 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: 0.25
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bwr
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30234.png

Downloads & links

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Map

FileDownload / File: emd_30234.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhalf map B from cryoSPARC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 256 pix.
= 209.92 Å		0.82 Å/pix.
x 256 pix.
= 209.92 Å		0.82 Å/pix.
x 256 pix.
= 209.92 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-1.0558376 - 2.0626483
Average (Standard dev.)0.006028593 (±0.09724904)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 209.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z209.920209.920209.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-1.0562.0630.006

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Supplemental data

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Mask #1

Fileemd_30234_msk_1.map
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Additional map: mask used in refinement from cryoSPARC

Fileemd_30234_additional.map
Annotationmask used in refinement from cryoSPARC
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Half map: half map A from cryoSPARC

Fileemd_30234_half_map_1.map
Annotationhalf map A from cryoSPARC
Projections & Slices
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Histogram

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Half map: half map B from cryoSPARC

Fileemd_30234_half_map_2.map
Annotationhalf map B from cryoSPARC
Projections & Slices
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Sample components

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Entire : Mycobacterial Arabinosyltransferase Complex EmbB2-AcpM2

EntireName: Mycobacterial Arabinosyltransferase Complex EmbB2-AcpM2
Components
  • Complex: Mycobacterial Arabinosyltransferase Complex EmbB2-AcpM2
    • Protein or peptide: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
    • Protein or peptide: Meromycolate extension acyl carrier protein
  • Ligand: CALCIUM ION
  • Ligand: [(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

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Supramolecule #1: Mycobacterial Arabinosyltransferase Complex EmbB2-AcpM2

SupramoleculeName: Mycobacterial Arabinosyltransferase Complex EmbB2-AcpM2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 254 kDa/nm

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Macromolecule #1: Integral membrane indolylacetylinositol arabinosyltransferase EmbB

MacromoleculeName: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: indolylacetylinositol arabinosyltransferase
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 116.870289 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MSGNMDEAVS GNMDEAVSAG KDVRIARWVA TIAGLLGFVL SVSIPLLPVT QTTATLNWPQ QGRLDNVTAP LISQAPLELT ATVPCSVVR DLPPEGGLVF GTAPAEGRDA ALNAMLVNVT ETRVDVIVRN VVVASVNRDR VAGPDCQRIE ITSNLDGTYA D FVGLTQIS ...String:
MSGNMDEAVS GNMDEAVSAG KDVRIARWVA TIAGLLGFVL SVSIPLLPVT QTTATLNWPQ QGRLDNVTAP LISQAPLELT ATVPCSVVR DLPPEGGLVF GTAPAEGRDA ALNAMLVNVT ETRVDVIVRN VVVASVNRDR VAGPDCQRIE ITSNLDGTYA D FVGLTQIS GEDAGKLQRT GYPDPNLRPA IVGVFTDLTG PAPQGLSVSA EIDTRFTTHP TALKLAAMLL AIVSTVIALL AL WRLDRLD GRRMHRLIPT RWRTVTAVDG VVVGGMAIWY VIGANSSDDG YILQMARTAE HAGYMANYFR WFGSPEDPFG WYY NVLALM TKVSDASIWI RLPDLICALI CWLLLSREVL PRLGPAVAGS RAAMWAAGLV LLGAWMPFNN GLRPEGQIAT GALI TYVLI ERAVTSGRLT PAALAITTAA FTLGIQPTGL IAVAALLAGG RPILRIVMRR RRLVGTWPLI APLLAAGTVI LAVVF ADQT IATVLEATRI RTAIGPSQEW WTENLRYYYL ILPTTDGAIS RRVAFVFTAM CLFPSLFMML RRKHIAGVAR GPAWRL MGI IFATMFFLMF TPTKWIHHFG LFAAVGGAMA ALATVLVSPT VLRSARNRMA FLSLVLFVLA FCFASTNGWW YVSNFGA PF NNSVPKVGGV QISAIFFALS AIAALWAFWL HLTRRTESRV VDRLTAAPIP VAAGFMVVVM MASMAIGVVR QYPTYSNG W ANIRAFAGGC GLADDVLVEP DSNAGFLTPL PGAYGPLGPL GGEDPQGFSP DGVPDRIIAE AIRLNNPQPG TDYDWNRPI KLDEPGINGS TVPLPYGLDP KRVPVAGTYS TEAQQESRLS SAWYELPARD ETERAAHPLV VITAAGTITG ESVANGLTTG QTVDLEYAT RGPDGTLVPA GRVTPYDVGP TPSWRNLRYP RSEIPDDAVA VRVVAEDLSL SQGDWIAVTP PRVPELQSVQ E YVGSDQPV LMDWAVGLAF PCQQPMLHAN GVTEVPKFRI SPDYYAKLQS TDTWQDGING GLLGITDLLL RASVMSTYLS QD WGQDWGS LRKFDTVVEA TPAELDFGSQ THSGLYSPGP LRIRP

UniProtKB: Integral membrane indolylacetylinositol arabinosyltransferase EmbB

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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
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString:
MAATQEEIIA GLAEIIEEVT GIEPSEVTPE KSFVDDLDID SLSMVEIAVQ TEDKYGVKIP DEDLAGLRTV GDVVAYIQKL EEENPEAAA ALREKFAADQ

UniProtKB: Meromycolate extension acyl carrier protein

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: [(2Z,6E,10E,14Z,18E,22Z,26Z)-3,7,11,15,19,23,27,31,35,39-decameth...

MacromoleculeName: [(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
type: ligand / ID: 4 / Number of copies: 1 / Formula: F8L
Molecular weightTheoretical: 911.28 Da
Chemical component information

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

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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

BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 125899
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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