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- PDB-8dky: Crystal structure of the Aquifex aeolicus Wzt Carbohydrate Bindin... -

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Basic information

Entry
Database: PDB / ID: 8dky
TitleCrystal structure of the Aquifex aeolicus Wzt Carbohydrate Binding Domain bound to 3-O-methyl-D-mannose
ComponentsABC transporter
KeywordsSUGAR BINDING PROTEIN / O antigen ABC transporter / carbohydrate binding domain / 3-O-methyl-D-mannose
Function / homology
Function and homology information


ABC-type transporter activity / ATP hydrolysis activity / ATP binding / membrane
Similarity search - Function
Wzt, C-terminal / Wzt C-terminal domain / ABC transporter, teichoic acids export TagH-like / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
3-O-methyl-alpha-D-mannopyranose / ABC transporter
Similarity search - Component
Biological speciesAquifex aeolicus VF5 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.61 Å
AuthorsSpellmon, N. / Zimmer, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Commun / Year: 2022
Title: Molecular basis for polysaccharide recognition and modulated ATP hydrolysis by the O antigen ABC transporter.
Authors: Nicholas Spellmon / Artur Muszyński / Ireneusz Górniak / Jiri Vlach / David Hahn / Parastoo Azadi / Jochen Zimmer /
Abstract: O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked ...O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.
History
DepositionJul 6, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ABC transporter
B: ABC transporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,6754
Polymers33,2862
Non-polymers3882
Water2,684149
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4500 Å2
ΔGint-34 kcal/mol
Surface area13550 Å2
MethodPISA
Unit cell
Length a, b, c (Å)56.561, 58.169, 95.316
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein ABC transporter


Mass: 16643.156 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus VF5 (bacteria) / Strain: VF5 / Gene: abcT4, aq_1094 / Production host: Escherichia coli (E. coli) / References: UniProt: O67181
#2: Chemical ChemComp-U90 / 3-O-methyl-alpha-D-mannopyranose


Mass: 194.182 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C7H14O6 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 149 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.36 Å3/Da / Density % sol: 47.78 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8.8 / Details: 20% PEG 3350, 0.1M Tricine pH 8.8

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 23, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.61→95.32 Å / Num. obs: 41212 / % possible obs: 99.3 % / Redundancy: 6.8 % / Rmerge(I) obs: 0.087 / Rpim(I) all: 0.037 / Net I/σ(I): 12
Reflection shellResolution: 1.61→1.64 Å / Redundancy: 5.6 % / Rmerge(I) obs: 0.782 / Mean I/σ(I) obs: 2 / Num. unique obs: 1844 / CC1/2: 0.809 / Rpim(I) all: 0.355 / % possible all: 91.7

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
PDB_EXTRACT3.27data extraction
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6O14

6o14


Resolution: 1.61→48.64 Å / SU ML: 0.22 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 34.08 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2875 2126 5.17 %
Rwork0.2598 38968 -
obs0.2612 41094 99.11 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 54 Å2 / Biso mean: 26.3494 Å2 / Biso min: 11.83 Å2
Refinement stepCycle: final / Resolution: 1.61→48.64 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2344 0 26 151 2521
Biso mean--25.42 31.63 -
Num. residues----288
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 15

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.61-1.650.43981280.39362392252093
1.65-1.690.3811260.345525682694100
1.69-1.730.34561290.319825972726100
1.73-1.790.35031580.307125772735100
1.79-1.840.32481380.302825882726100
1.84-1.910.30511370.282325772714100
1.91-1.990.29641580.26562592275099
1.99-2.080.28331320.25612535266798
2.08-2.190.29261490.259626162765100
2.19-2.320.30141550.262525972752100
2.32-2.50.29281360.270625902726100
2.5-2.750.30721570.273626212778100
2.75-3.150.27131390.26622636277599
3.15-3.970.25061390.236226792818100
3.97-48.640.26021450.222628032948100

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