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- PDB-6w98: Single-Particle Cryo-EM Structure of Arabinofuranosyltransferase ... -

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Entry
Database: PDB / ID: 6w98
TitleSingle-Particle Cryo-EM Structure of Arabinofuranosyltransferase AftD from Mycobacteria
Components
  • Acyl carrier protein
  • F5/8 type C domain-containing protein
KeywordsMEMBRANE PROTEIN / Glycosyltransferase / lipomannan / lipoarabinomannan / arabinofuranose / nanodisc / single-particle cryo-electron microscopy / acyl carrier protein
Function / homology
Function and homology information


lipid A biosynthetic process / lipid biosynthetic process / acyl binding / acyl carrier activity / phosphopantetheine binding / membrane => GO:0016020 / fatty acid biosynthetic process / transferase activity / response to xenobiotic stimulus / lipid binding ...lipid A biosynthetic process / lipid biosynthetic process / acyl binding / acyl carrier activity / phosphopantetheine binding / membrane => GO:0016020 / fatty acid biosynthetic process / transferase activity / response to xenobiotic stimulus / lipid binding / membrane / cytosol / cytoplasm
Similarity search - Function
Alpha-(1->3)-arabinofuranosyltransferase / Alpha-(1->3)-arabinofuranosyltransferase, N-terminal GT-C domain / Coagulation factors 5/8 type C domain (FA58C) profile. / Coagulation factor 5/8 C-terminal domain / Acyl carrier protein (ACP) / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Galactose-binding-like domain superfamily ...Alpha-(1->3)-arabinofuranosyltransferase / Alpha-(1->3)-arabinofuranosyltransferase, N-terminal GT-C domain / Coagulation factors 5/8 type C domain (FA58C) profile. / Coagulation factor 5/8 C-terminal domain / Acyl carrier protein (ACP) / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Galactose-binding-like domain superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain
Similarity search - Domain/homology
Chem-6OU / 4'-PHOSPHOPANTETHEINE / F5/8 type C domain-containing protein / Acyl carrier protein
Similarity search - Component
Biological speciesMycobacteroides abscessus subsp. abscessus (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 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 22, 2020Deposition site: RCSB / Processing site: RCSB
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Assembly

Deposited unit
A: F5/8 type C domain-containing protein
B: Acyl carrier protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,3866
Polymers158,2302
Non-polymers1,1574
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Wild-type Mycobacterial Arabinofuranosyltransferase AftD Complexed with Acyl Carrier Protein
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1620 Å2
ΔGint-12 kcal/mol
Surface area54940 Å2

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein F5/8 type C domain-containing protein


Mass: 149584.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacteroides abscessus subsp. abscessus (bacteria)
Gene: SAMEA2161603_00457 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1N1NKH9
#2: Protein Acyl carrier protein / ACP / Cytosolic-activating factor / CAF / Fatty acid synthase acyl carrier protein


Mass: 8645.460 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / Strain: K12 / References: UniProt: P0A6A8

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Non-polymers , 4 types, 5 molecules

#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-6OU / [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate


Mass: 717.996 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C39H76NO8P / Comment: phospholipid*YM
#5: Chemical ChemComp-PNS / 4'-PHOSPHOPANTETHEINE


Mass: 358.348 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H23N2O7PS / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Wild-type Mycobacterial Arabinofuranosyltransferase AftD Complexed with Acyl Carrier ProteinORGANELLE OR CELLULAR COMPONENT#1-#20MULTIPLE SOURCES
2F5/8 type C domain-containing proteinCOMPLEX#11RECOMBINANT
3Acyl carrier proteinORGANELLE OR CELLULAR COMPONENT#21NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.16 MDaNO
210.049 MDaNO
310.050 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mycobacteroides abscessus subsp. abscessus (bacteria)1185650
23Escherichia coli (strain K12) (bacteria)83333
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 ChlorideNaCl1
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: COPPER / Grid type: Homemade
VitrificationInstrument: SPOTITON / Cryogen name: ETHANE / Chamber temperature: 298 K
Details: The Spotiton V1.0 robot was operating at room temperature and moderate humidity.

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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 FIELD / 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: 95.72 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 7274
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
1Gautomatch0.53particle selection
2Leginon3.4image acquisition
4RELION2.1CTF correction
5cisTEM1CTF correction
6cryoSPARC2CTF correction
9Cootmodel fitting
10Buccaneermodel fitting
12PHENIXmodel refinement
13cryoSPARC2initial Euler assignment
14cryoSPARC2final Euler assignment
15RELION2.1classification
16RELION2.13D reconstruction
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: 2800102
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 490616 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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