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- PDB-6xfu: PmtCD peptide exporter basket domain -

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Basic information

Entry
Database: PDB / ID: 6xfu
TitlePmtCD peptide exporter basket domain
ComponentsABC transporter ATP-binding protein
KeywordsMEMBRANE PROTEIN / ABC transporter / ABC exporter / Peptide transort
Function / homology
Function and homology information


ATP hydrolysis activity / ATP binding
Similarity search - Function
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
THIOCYANATE ION / ABC transporter, ATP-binding protein, putative / ABC transporter ATP-binding protein
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.4 Å
AuthorsZeytuni, N. / Strynadka, N.C.J. / Alexander, J.A.N.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)Operating grant Canada
CitationJournal: Sci Adv / Year: 2020
Title: Structural insight into the ATP-driven exporter of virulent peptide toxins.
Authors: N Zeytuni / S W Dickey / J Hu / H T Chou / L J Worrall / J A N Alexander / M L Carlson / M Nosella / F Duong / Z Yu / M Otto / N C J Strynadka /
Abstract: is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs ... is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an "open" conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.
History
DepositionJun 16, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 14, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 5, 2023Group: Database references / Category: database_2 / pdbx_database_related
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 25, 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 ATP-binding protein
B: ABC transporter ATP-binding protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,4494
Polymers16,3322
Non-polymers1162
Water3,027168
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1690 Å2
ΔGint-14 kcal/mol
Surface area8300 Å2
MethodPISA
Unit cell
Length a, b, c (Å)40.462, 45.375, 73.820
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP22121
Components on special symmetry positions
IDModelComponents
11A-458-

HOH

21A-479-

HOH

31B-425-

HOH

41B-473-

HOH

51B-487-

HOH

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Components

#1: Protein ABC transporter ATP-binding protein / ABC transporter / ATP-binding protein / Antibiotic ABC transporter ATP-binding protein / Antibiotic ...ABC transporter / ATP-binding protein / Antibiotic ABC transporter ATP-binding protein / Antibiotic transport system ATP-binding protein / Phenol-soluble modulin export ABC transporter ATP-binding protein PmtC


Mass: 8166.194 Da / Num. of mol.: 2 / Fragment: basket domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria)
Gene: ybhF_2, pmtC, ybhF_1, ybhF_3, BN1321_320024, BTN44_02050, C7P97_04805, CSC83_02360, CSC87_13000, EP54_10395, EQ90_13985, ER624_12880, ERS072840_01916, HMPREF3211_02244, NCTC10654_02103, ...Gene: ybhF_2, pmtC, ybhF_1, ybhF_3, BN1321_320024, BTN44_02050, C7P97_04805, CSC83_02360, CSC87_13000, EP54_10395, EQ90_13985, ER624_12880, ERS072840_01916, HMPREF3211_02244, NCTC10654_02103, NCTC13131_01274, NCTC7878_02640, NCTC7988_02070, RK64_10625
Production host: Escherichia coli BL21 (bacteria) / References: UniProt: X5EJW5, UniProt: Q2FWW9*PLUS
#2: Chemical ChemComp-SCN / THIOCYANATE ION


Mass: 58.082 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: CNS / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 168 / 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: 2.09 Å3/Da / Density % sol: 41.03 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 7.4
Details: 0.2 M sodium thiocyanate and 20% Poly Ethylene Glycol (PEG) 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 1.033 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: May 30, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.033 Å / Relative weight: 1
ReflectionResolution: 1.4→45.37 Å / Num. obs: 27091 / % possible obs: 98.5 % / Redundancy: 11.6 % / CC1/2: 0.998 / Rmerge(I) obs: 0.098 / Rpim(I) all: 0.029 / Rrim(I) all: 0.103 / Net I/σ(I): 14.7 / Num. measured all: 314139 / Scaling rejects: 37
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
1.4-1.4260.653688911520.7590.2870.7172.185.4
7.67-45.37100.06921262130.9980.0220.07327.799.7

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
Aimless0.7.4data scaling
PHASERphasing
PDB_EXTRACT3.25data extraction
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6U2D

6u2d


Resolution: 1.4→40.46 Å / Cor.coef. Fo:Fc: 0.974 / Cor.coef. Fo:Fc free: 0.965 / WRfactor Rfree: 0.2086 / WRfactor Rwork: 0.1699 / FOM work R set: 0.8736 / SU B: 0.992 / SU ML: 0.039 / SU R Cruickshank DPI: 0.0598 / SU Rfree: 0.0639 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.06 / ESU R Free: 0.064 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1937 1378 5.1 %RANDOM
Rwork0.1609 ---
obs0.1625 25672 98.39 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 54.13 Å2 / Biso mean: 18.273 Å2 / Biso min: 10.54 Å2
Baniso -1Baniso -2Baniso -3
1--0.97 Å20 Å2-0 Å2
2--0.44 Å20 Å2
3---0.53 Å2
Refinement stepCycle: final / Resolution: 1.4→40.46 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1140 0 6 168 1314
Biso mean--20.64 31.07 -
Num. residues----139
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.0131220
X-RAY DIFFRACTIONr_bond_other_d0.0010.0171119
X-RAY DIFFRACTIONr_angle_refined_deg1.8711.6431655
X-RAY DIFFRACTIONr_angle_other_deg1.5211.592623
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2015153
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.35224.47867
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.06315235
X-RAY DIFFRACTIONr_dihedral_angle_4_deg27.161154
X-RAY DIFFRACTIONr_chiral_restr0.0870.2166
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.021353
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02235
LS refinement shellResolution: 1.4→1.436 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.25 82 -
Rwork0.226 1615 -
all-1697 -
obs--85.79 %

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