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- PDB-6wbx: Single-Particle Cryo-EM Structure of Arabinofuranosyltransferase ... -

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Basic information

Entry
Database: PDB / ID: 6wbx
TitleSingle-Particle Cryo-EM Structure of Arabinofuranosyltransferase AftD from Mycobacteria, Mutant R1389S Class 1
ComponentsDUF3367 domain-containing protein
KeywordsMEMBRANE PROTEIN / Glycosyltransferase / 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 => GO:0016020 / DUF3367 domain-containing protein
Function and homology information
Biological speciesMycobacteroides abscessus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsTan, Y.Z. / Zhang, L. / Rodrigues, J. / Zheng, R.B. / Giacometti, S.I. / Rosario, A.L. / Kloss, B. / Dandey, V.P. / Wei, H. / Brunton, R. ...Tan, Y.Z. / Zhang, L. / Rodrigues, J. / Zheng, R.B. / Giacometti, S.I. / Rosario, A.L. / Kloss, B. / Dandey, V.P. / Wei, H. / Brunton, R. / Raczkowski, A.M. / Athayde, D. / Catalao, M.J. / Pimentel, M. / Clarke, O.B. / Lowary, T.L. / Archer, M. / Niederweis, M. / Potter, C.S. / Carragher, B. / Mancia, F.
Funding support United States, 6items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 13, 2020Provider: repository / Type: Initial release
Revision 1.1May 20, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jun 3, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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Assembly

Deposited unit
A: DUF3367 domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,8993
Polymers152,8191
Non-polymers802
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area49590 Å2

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Components

#1: Protein DUF3367 domain-containing protein


Mass: 152818.641 Da / Num. of mol.: 1 / Mutation: R1389S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacteroides abscessus (bacteria) / Gene: D2E76_26050 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A418KZ72
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Mutant R1389S Class 1 Mycobacterial Arabinofuranosyltransferase AftD Complexed with Acyl Carrier Protein
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.15 MDaNO
211 MDaNO
315 MDaNO
Source (natural)Organism: Mycobacteroides abscessus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: Solution was filtered and degassed.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2200 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Protein was incorporated into lipid nanodiscs.
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 13.05 sec. / Electron dose: 96.78 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4886
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 15 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 90 / Used frames/image: 1-90

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Processing

EM software
IDNameVersionCategory
2Leginon3.4image acquisition
4RELION2.1CTF correction
5cisTEM1CTF correction
6cryoSPARC2CTF correction
9Cootmodel fitting
11cryoSPARC2initial Euler assignment
12cryoSPARC2final Euler assignment
13RELION2.1classification
14RELION2.13D reconstruction
15PHENIXmodel refinement
Image processingDetails: Energy filter slit width of 15 eV was used during the collection and was aligned automatically every hour using Leginon.
CTF correctionDetails: CTF was estimated using GCTF and refined throughout the pipeline using cisTEM.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 226478
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37814 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6W98

6w98


Pdb chain-ID: A

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