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- EMDB-21983: Single-Particle Cryo-EM Structure of Arabinosyltransferase EmbB f... -

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Basic information

Entry
Database: EMDB / ID: EMD-21983
TitleSingle-Particle Cryo-EM Structure of Arabinosyltransferase EmbB from Mycobacterium smegmatis
Map dataDensity modified map
Sample
  • Organelle or cellular component: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
    • Protein or peptide: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
  • Ligand: CALCIUM IONCalcium
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
KeywordsGlycosyltransferase / membrane protein / 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
Probable arabinosyltransferase A / Integral membrane indolylacetylinositol arabinosyltransferase EmbB
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (bacteria) / Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsTan YZ / Rodrigues J
Funding support United States, Portugal, European Union, Canada, 12 items
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, 2020-
Header (metadata) releaseJun 10, 2020-
Map releaseJun 10, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.325
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.325
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6x0o
  • Surface level: 0.325
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21983.png

Downloads & links

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Map

FileDownload / File: emd_21983.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity modified map
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.325 / Movie #1: 0.325
Minimum - Maximum-1.0804 - 2.3828406
Average (Standard dev.)0.000000000000439 (±0.04592852)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 256.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z256.000256.000256.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-1.0802.3830.000

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Supplemental data

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Mask #1

Fileemd_21983_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Raw map

Fileemd_21983_additional_1.map
AnnotationRaw map
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AxesZYX

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Additional map: CryoSPARC Sharpened Map

Fileemd_21983_additional_2.map
AnnotationCryoSPARC Sharpened Map
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Additional map: 3DFSC - Raw

Fileemd_21983_additional_3.map
Annotation3DFSC - Raw
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Additional map: 3DFSC - Thresholded

Fileemd_21983_additional_4.map
Annotation3DFSC - Thresholded
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Additional map: 3DFSC - Thresholded and binarized

Fileemd_21983_additional_5.map
Annotation3DFSC - Thresholded and binarized
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Additional map: Local resolution map

Fileemd_21983_additional_6.map
AnnotationLocal resolution map
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Half map: Half map 1

Fileemd_21983_half_map_1.map
AnnotationHalf map 1
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Half map: Half map 2

Fileemd_21983_half_map_2.map
AnnotationHalf map 2
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Sample components

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Entire : Integral membrane indolylacetylinositol arabinosyltransferase EmbB

EntireName: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
Components
  • Organelle or cellular component: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
    • Protein or peptide: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
  • Ligand: CALCIUM IONCalcium
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE

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Supramolecule #1: Integral membrane indolylacetylinositol arabinosyltransferase EmbB

SupramoleculeName: Integral membrane indolylacetylinositol arabinosyltransferase EmbB
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 119 KDa

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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: 1 / 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: 118.835375 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 LRIRPAENLY FQGWSHPQFE K

UniProtKB: Integral membrane indolylacetylinositol arabinosyltransferase EmbB

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Macromolecule #2: CALCIUM ION

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

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Macromolecule #3: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE

MacromoleculeName: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / type: ligand / ID: 3 / Number of copies: 2 / Formula: LHG
Molecular weightTheoretical: 722.97 Da
Chemical component information

ChemComp-LHG:
1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / phospholipid*YM / Phosphatidylglycerol

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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

Concentration8 mg/mL
BufferpH: 7.5
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 278 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: FEI FALCON III (4k x 4k) / #0 - Detector mode: COUNTING / #0 - Number grids imaged: 1 / #0 - Number real images: 2158 / #0 - Average exposure time: 86.4 sec. / #0 - Average electron dose: 78.02 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Detector mode: COUNTING / #1 - Digitization - Dimensions - Width: 3838 pixel / #1 - Digitization - Dimensions - Height: 3710 pixel / #1 - Number grids imaged: 1 / #1 - Number real images: 7833 / #1 - Average exposure time: 8.0 sec. / #1 - Average electron dose: 77.53 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing #1

Particle selectionNumber selected: 162271
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model / Details: Ab initio model was generated in CryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2) / Details: Non-uniform refinement in CryoSPARC was used.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2) / Details: Non-uniform refinement in CryoSPARC was used.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Details: Combined with K2 dataset / Number images used: 57970
Image processing ID1
Image recording ID1
FSC plot (resolution estimation)

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Image processing #2

Particle selectionNumber selected: 700201
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model / Details: Ab initio model was generated in CryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2) / Details: Non-uniform refinement in CryoSPARC was used.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2) / Details: Non-uniform refinement in CryoSPARC was used.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Details: Combined with Falcon III dataset / Number images used: 57970
Image processing ID2
Image recording ID2
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3pty


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: AB INITIO MODEL
Output model

PDB-6x0o:
Single-Particle Cryo-EM Structure of Arabinosyltransferase EmbB from Mycobacterium smegmatis

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