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- PDB-8f7h: The condensation domain of surfactin A synthetase C variant 18b i... -

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Basic information

Entry
Database: PDB / ID: 8f7h
TitleThe condensation domain of surfactin A synthetase C variant 18b in space group P212121
ComponentsSurfactin synthetase
KeywordsBIOSYNTHETIC PROTEIN / NRPS / C domain / SrfA-C
Function / homology
Function and homology information


amino acid activation for nonribosomal peptide biosynthetic process / secondary metabolite biosynthetic process / lipid biosynthetic process / catalytic activity / phosphopantetheine binding / cytosol
Similarity search - Function
Condensation domain / Condensation domain / AMP-binding, conserved site / Putative AMP-binding domain signature. / Chloramphenicol acetyltransferase-like domain superfamily / AMP-dependent synthetase/ligase / AMP-binding enzyme
Similarity search - Domain/homology
Surfactin synthetase
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.93 Å
AuthorsFrota, N.F. / Pistofidis, A. / Folger, I.B. / Hilvert, D. / Schmeing, T.M.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)178084 Canada
CitationJournal: Nat Chem Biol / Year: 2024
Title: High-throughput reprogramming of an NRPS condensation domain.
Authors: Ines B Folger / Natália F Frota / Angelos Pistofidis / David L Niquille / Douglas A Hansen / T Martin Schmeing / Donald Hilvert /
Abstract: Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogs. Although yeast display is a proven, powerful tool for altering the ...Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogs. Although yeast display is a proven, powerful tool for altering the substrate specificity of gatekeeper adenylation domains in nonribosomal peptide synthetases (NRPSs), comparable strategies for other components of these megaenzymes have not been described. Here we report a high-throughput approach for engineering condensation (C) domains responsible for peptide elongation. We show that a 120-kDa NRPS module, displayed in functional form on yeast, can productively interact with an upstream module, provided in solution, to produce amide products tethered to the yeast surface. Using this system to screen a large C-domain library, we reprogrammed a surfactin synthetase module to accept a fatty acid donor, increasing catalytic efficiency for this noncanonical substrate >40-fold. Because C domains can function as selectivity filters in NRPSs, this methodology should facilitate the precision engineering of these molecular assembly lines.
History
DepositionNov 18, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 22, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / citation_author
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 / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 12, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Feb 4, 2026Group: Structure summary / Category: audit_author / pdbx_entry_details
Item: _audit_author.name / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Surfactin synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,6722
Polymers53,5801
Non-polymers921
Water2,918162
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)75.736, 83.722, 86.379
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 Surfactin synthetase / Surfactin A synthetase C


Mass: 53579.785 Da / Num. of mol.: 1 / Fragment: Condensation domain, residues 7-441
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q45676
#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 162 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: 2.56 Å3/Da / Density % sol: 51.87 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, sitting drop
Details: 0.04 M KH2PO4, 16 %w/v PEG 8K and 20% (v/v) glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 19, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 1.9→50 Å / Num. obs: 43869 / % possible obs: 100 % / Redundancy: 13.2 % / Biso Wilson estimate: 44.46 Å2 / CC1/2: 0.998 / Net I/σ(I): 8.4
Reflection shellResolution: 1.9→1.93 Å / Redundancy: 13.5 % / Num. unique obs: 2156 / CC1/2: 0.39 / % possible all: 100

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Processing

Software
NameVersionClassification
HKL-2000data collection
HKL-2000data scaling
HKL-2000data reduction
PHASERphasing
PHENIX1.19.2_4158refinement
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2VSQ

2vsq


Resolution: 1.93→47.09 Å / SU ML: 0.2303 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 23.217
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2278 1999 4.78 %
Rwork0.1972 39863 -
obs0.1986 41862 98.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 58.86 Å2
Refinement stepCycle: LAST / Resolution: 1.93→47.09 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3512 0 6 162 3680
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01513609
X-RAY DIFFRACTIONf_angle_d1.26394882
X-RAY DIFFRACTIONf_chiral_restr0.0722536
X-RAY DIFFRACTIONf_plane_restr0.0118631
X-RAY DIFFRACTIONf_dihedral_angle_d5.3553467
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.93-1.970.32971280.30522619X-RAY DIFFRACTION91.84
1.97-2.030.33121430.27382764X-RAY DIFFRACTION98.34
2.03-2.090.30541400.25712823X-RAY DIFFRACTION98.83
2.09-2.150.26651450.25272799X-RAY DIFFRACTION98.96
2.15-2.230.31891290.24462821X-RAY DIFFRACTION99.19
2.23-2.320.26821450.22732824X-RAY DIFFRACTION99.6
2.32-2.430.23941420.20982836X-RAY DIFFRACTION99.57
2.43-2.550.26561450.21562865X-RAY DIFFRACTION99.7
2.55-2.710.2221450.20532862X-RAY DIFFRACTION99.87
2.71-2.920.25591440.22022854X-RAY DIFFRACTION99.83
2.92-3.220.24521440.21632896X-RAY DIFFRACTION99.9
3.22-3.680.25911480.20232897X-RAY DIFFRACTION99.97
3.68-4.640.19491480.1692926X-RAY DIFFRACTION100
4.64-47.090.18651530.17163077X-RAY DIFFRACTION99.85
Refinement TLS params.Method: refined / Origin x: 1.44722901559 Å / Origin y: -4.99347587703 Å / Origin z: -5.01156963979 Å
111213212223313233
T0.283463268447 Å2-0.0616224376354 Å2-0.00352627034954 Å2-0.311355857792 Å20.0144885541599 Å2--0.227660118013 Å2
L1.8494167832 °2-0.604805471867 °2-0.468242418232 °2-2.32652611184 °2-0.164092219301 °2--1.29117190719 °2
S0.119377495351 Å °-0.0586115719044 Å °0.183723343449 Å °-0.104356500739 Å °-0.0594772631587 Å °-0.0422502310333 Å °-0.341193140209 Å °0.143729184192 Å °-0.0426894605775 Å °
Refinement TLS groupSelection details: all

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