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- EMDB-21601: Single-Particle Cryo-EM Structure of Arabinofuranosyltransferase ... -

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Entry
Database: EMDB / ID: EMD-21601
TitleSingle-Particle Cryo-EM Structure of Arabinofuranosyltransferase AftD from Mycobacteria, Mutant R1389S Class 2
Map dataSharpened map
Sample
  • Organelle or cellular component: Mutant R1389S Class 2 Mycobacterial Arabinofuranosyltransferase AftD Complexed with Acyl Carrier Protein
    • Protein or peptide: DUF3367 domain-containing protein
  • Ligand: CALCIUM ION
KeywordsGlycosyltransferase / membrane protein / nanodisc / acyl carrier protein
Function / homologyAlpha-(1->3)-arabinofuranosyltransferase / Alpha-(1->3)-arabinofuranosyltransferase / Coagulation factors 5/8 type C domain (FA58C) profile. / Coagulation factor 5/8 C-terminal domain / Galactose-binding-like domain superfamily / transferase activity / membrane / DUF3367 domain-containing protein
Function and homology information
Biological speciesMycobacteroides abscessus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsTan YZ / Zhang L
Funding support United States, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103310 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 GM116799 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103310 United States
CitationJournal: Mol Cell / Year: 2020
Title: Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria.
Authors: Yong Zi Tan / Lei Zhang / José Rodrigues / Ruixiang Blake Zheng / Sabrina I Giacometti / Ana L Rosário / Brian Kloss / Venkata P Dandey / Hui Wei / Richard Brunton / Ashleigh M Raczkowski ...Authors: Yong Zi Tan / Lei Zhang / José Rodrigues / Ruixiang Blake Zheng / Sabrina I Giacometti / Ana L Rosário / Brian Kloss / Venkata P Dandey / Hui Wei / Richard Brunton / Ashleigh M Raczkowski / Diogo Athayde / Maria João Catalão / Madalena Pimentel / Oliver B Clarke / Todd L Lowary / Margarida Archer / Michael Niederweis / Clinton S Potter / Bridget Carragher / Filippo Mancia /
Abstract: Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two ...Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function.
History
DepositionMar 27, 2020-
Header (metadata) releaseMay 13, 2020-
Map releaseMay 13, 2020-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 6.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wby
  • Surface level: 6.5
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21601.jpg

emd_21601.png

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Map

FileDownload / File: emd_21601.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.488 Å		1.06 Å/pix.
x 256 pix.
= 271.488 Å		1.06 Å/pix.
x 256 pix.
= 271.488 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0605 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.5 / Movie #1: 6.5
Minimum - Maximum-29.877168999999999 - 47.496406999999998
Average (Standard dev.)-0.000000000006393 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.488 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06051.06051.0605
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.488271.488271.488
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-29.87747.496-0.000

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Supplemental data

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Mask #1

Fileemd_21601_msk_1.map
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Additional map: Raw map

Fileemd_21601_additional_1.map
AnnotationRaw map
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Additional map: Local resolution map

Fileemd_21601_additional_2.map
AnnotationLocal resolution map
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Additional map: 3DFSC

Fileemd_21601_additional_3.map
Annotation3DFSC
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Additional map: 3DFSC - Thresholded

Fileemd_21601_additional_4.map
Annotation3DFSC - Thresholded
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Additional map: 3DFSC - Thresholded, Binarized

Fileemd_21601_additional_5.map
Annotation3DFSC - Thresholded, Binarized
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Half map: Half map 2

Fileemd_21601_half_map_1.map
AnnotationHalf map 2
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Half map: Half map 1

Fileemd_21601_half_map_2.map
AnnotationHalf map 1
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Sample components

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Entire : Mutant R1389S Class 2 Mycobacterial Arabinofuranosyltransferase A...

EntireName: Mutant R1389S Class 2 Mycobacterial Arabinofuranosyltransferase AftD Complexed with Acyl Carrier Protein
Components
  • Organelle or cellular component: Mutant R1389S Class 2 Mycobacterial Arabinofuranosyltransferase AftD Complexed with Acyl Carrier Protein
    • Protein or peptide: DUF3367 domain-containing protein
  • Ligand: CALCIUM ION

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Supramolecule #1: Mutant R1389S Class 2 Mycobacterial Arabinofuranosyltransferase A...

SupramoleculeName: Mutant R1389S Class 2 Mycobacterial Arabinofuranosyltransferase AftD Complexed with Acyl Carrier Protein
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mycobacteroides abscessus (bacteria)
Molecular weightTheoretical: 5 MDa

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Macromolecule #1: DUF3367 domain-containing protein

MacromoleculeName: DUF3367 domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacteroides abscessus (bacteria)
Molecular weightTheoretical: 152.818641 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDYKDDDDKH HHHHHHHHHE NLYFQSYVMT YRLDSSALSR RWLAVAAAVS LLLTFSQSPG QISPDTKLDL AINPLRFAAR ALNLWSSDL PFGQAQNQAY GYLFPHGAFF SLGHLLGVPA WVTQRLWWAL LIVAGFWGLI RVAEALGIGT RGSRIIAAVA F ALSPRVLT ...String:
MDYKDDDDKH HHHHHHHHHE NLYFQSYVMT YRLDSSALSR RWLAVAAAVS LLLTFSQSPG QISPDTKLDL AINPLRFAAR ALNLWSSDL PFGQAQNQAY GYLFPHGAFF SLGHLLGVPA WVTQRLWWAL LIVAGFWGLI RVAEALGIGT RGSRIIAAVA F ALSPRVLT TLGAISSETL PMMLAPWVLL PLILTFQGRM SPRRAAALSA VAVALMGAVN AVATALACGV AVIWWLAHRP NR TWWRFTA WWIPCLALAS TWWIVALLIF GKISPKFLDF IESSGVTTQW TSLTEVLRGT DSWTPFVAPT ATAGSSLVTQ SAM VIATTM LAAAGMAGLA MRGMPARGRL VAVLLIGLVL LTAGYTGALG SPIAQQIQFF LDDGGTPLRN VHKLEPLIRL PLIL GLAHA LSRIPLPASV PVRQWLSALA RPERNRAVAF AIVLLVALAA STSLAWTGRL VPRGGFDAIP GYWNDTAHWL ADHDT GGRA LVVPGAPFAI QTWGLTRDEP LQALGQTPWG VRDSIPLTPP ETIRAIDSVQ QLFAAGRPSD GLADTLREQG ISYLVV RND LDPDTSRSAR PILVHHTIEG SPGLTKVAQF GDPVGAGAVE GFVADSDLRP QYPAVEIYAV GANDHDGEPY FTDIDTM PR VAGGPEALLR LNERRRQLNE PPLGPSLLAT DAAQAGLRPG PAVVTDTPLA RETDYGRVDD HSSAIRAPGD KRRTFNRV P DYPATGVPLV NGSWTGGTIT ASSSASDSTA LPNVAPGTST AAAIDRDNAT SWVSSSLEAA LGQWIRIDLD RPITNAILT VTPSATALGA QVRRLEVETD NGTTSVRFDE PGQPLNIALR PGETTWVKVT ATGTDDGTSG VQFGVTELSL TQYDAAGFAH TVDLRHSAT VPPPPAGDNP LGWDLGSPLQ GRSGCAPSPQ RLRCAATLSL APEEPGTFIR TLTVPQPVSL TPRLWVRARP G PQLRDLIQ QPGTTVATGD SDVIDPQGSS YAATDGDPGT VWTAPQDSVQ RLHLPSLVIK LPKPTAIGAI RLRPSRTEVP AH PKQVAIN LGDGPQLRSI DPKADVTELA LHPSITDTIT VTVTDWTDII DRTALGFDQL KPPGIAEVIA LDADHRPIAP ADN AANSKR KITIGCNRGP ILALAGRFVP MSITATVREL LDGTVIQATP CDTSPIATGA GIQDVTVNPS QQFIVDGVQL TAAA TEPAS ATMTVAPKGA WGPDRREVTA EPSAHERVLA VPESINPGWA ARDAQGHLLT PVRVNGWQQG WVLPAGDGGK ITLTF GLNT WYRAGLFGGL ALLPILACLA LLPARGRTTL PPVAPWCAGP AAGVAVLAAL TAISGISGMA VGLAALAFKV WTRWPL RAV TAAGVYLAGG SLLLAGAALS RHPWRSVGGY TGHSWWIQLL ALISVASVAL AAVSLPSRRC WKRRSASREG DSTSA

UniProtKB: DUF3367 domain-containing protein

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
200.0 mMNaClSodium Chloride

Details: Solution was filtered and degassed.
GridModel: UltrAuFoil / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber temperature: 298 K / Instrument: LEICA EM GP
DetailsProtein was incorporated into lipid nanodiscs.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-90 / Number grids imaged: 1 / Number real images: 4886 / Average exposure time: 13.05 sec. / Average electron dose: 96.78 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
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

DetailsEnergy filter slit width of 15 eV was used during the collection and was aligned automatically every hour using Leginon.
Particle selectionNumber selected: 226478
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model / Details: Ab initio model was generated in CryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 68231
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 3D classificationNumber classes: 5 / Avg.num./class: 30196 / Software - Name: RELION (ver. 2.1)
Details: RELION 3D Focused Classification after Signal Subtraction
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6w98


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6wby:
Single-Particle Cryo-EM Structure of Arabinofuranosyltransferase AftD from Mycobacteria, Mutant R1389S Class 2

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