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- PDB-8ctq: Crystal structure of engineered phospholipase D mutant superPLD 2-48 -

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Basic information

Entry
Database: PDB / ID: 8ctq
TitleCrystal structure of engineered phospholipase D mutant superPLD 2-48
ComponentsPhospholipase D
KeywordsHYDROLASE / Enzyme / phospholipase / PLD / phospholipid
Function / homology
Function and homology information


phosphatidyltransferase activity / cardiolipin biosynthetic process / phospholipase D / phospholipase D activity
Similarity search - Function
Phospholipase D-like domain / PLD-like domain / Phospholipase D. Active site motifs. / Phospholipase D/Transphosphatidylase / Phospholipase D phosphodiesterase active site profile.
Similarity search - Domain/homology
PHOSPHATE ION / Phospholipase D
Similarity search - Component
Biological speciesStreptomyces sp. PMF (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.85 Å
AuthorsTei, R. / Bagde, S.R. / Fromme, J.C. / Baskin, J.M.
Funding support United States, 5items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)CAREER CHE-1749919 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136258 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124165 United States
National Science Foundation (NSF, United States)DMR-1829070 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124166-01A1 United States
CitationJournal: Nat.Chem. / Year: 2023
Title: Activity-based directed evolution of a membrane editor in mammalian cells.
Authors: Tei, R. / Bagde, S.R. / Fromme, J.C. / Baskin, J.M.
History
DepositionMay 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 26, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 7, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 19, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.4Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Phospholipase D
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,8912
Polymers54,7961
Non-polymers951
Water5,116284
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area230 Å2
ΔGint-7 kcal/mol
Surface area17800 Å2
MethodPISA
Unit cell
Length a, b, c (Å)72.828, 73.254, 87.190
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein Phospholipase D


Mass: 54796.168 Da / Num. of mol.: 1
Mutation: K57R, A59V, K109R, P245A, V264I, G328S, G381V, G406S, G429D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces sp. PMF (bacteria) / Strain: PMF / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Rosetta 2 / References: UniProt: P84147, phospholipase D
#2: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 284 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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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: 2.01 Å3/Da / Density % sol: 39 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 4.4
Details: 21% PEG8000, 0.15 M lithium sulfate, citrate-NaOH, pH 4.4

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.9792 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 17, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 1.85→56.09 Å / Num. obs: 40025 / % possible obs: 99.15 % / Redundancy: 13.1 % / Biso Wilson estimate: 28.98 Å2 / CC1/2: 0.998 / Net I/σ(I): 13.55
Reflection shellResolution: 1.85→1.918 Å / Mean I/σ(I) obs: 2.35 / Num. unique obs: 3612 / CC1/2: 0.879

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XDSdata scaling
PHENIXphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1V0Y

1v0y


Resolution: 1.85→56.09 Å / SU ML: 0.1925 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 21.1422
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2174 1957 4.89 %
Rwork0.1729 38068 -
obs0.1751 40025 99.15 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 36.66 Å2
Refinement stepCycle: LAST / Resolution: 1.85→56.09 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3631 0 5 284 3920
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01023710
X-RAY DIFFRACTIONf_angle_d1.06865054
X-RAY DIFFRACTIONf_chiral_restr0.0619575
X-RAY DIFFRACTIONf_plane_restr0.0103657
X-RAY DIFFRACTIONf_dihedral_angle_d6.5433510
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.85-1.90.30431190.25642366X-RAY DIFFRACTION88.09
1.9-1.950.25841520.20992691X-RAY DIFFRACTION99.86
1.95-2.010.21011380.18582686X-RAY DIFFRACTION99.93
2.01-2.070.21631360.17722747X-RAY DIFFRACTION100
2.07-2.150.25391340.1862707X-RAY DIFFRACTION100
2.15-2.230.23281240.17532721X-RAY DIFFRACTION99.96
2.23-2.330.22691540.1682705X-RAY DIFFRACTION100
2.33-2.460.22481450.16682722X-RAY DIFFRACTION100
2.46-2.610.22131530.17442718X-RAY DIFFRACTION99.97
2.61-2.810.19211460.17562739X-RAY DIFFRACTION100
2.81-3.090.2461490.17772727X-RAY DIFFRACTION100
3.09-3.540.21471370.17342779X-RAY DIFFRACTION100
3.54-4.460.1621290.14862810X-RAY DIFFRACTION100
4.46-56.090.24121410.17832950X-RAY DIFFRACTION99.97

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