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- PDB-8f7g: The condensation domain of surfactin A synthetase C in space grou... -

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Basic information

Entry
Database: PDB / ID: 8f7g
TitleThe condensation domain of surfactin A synthetase C 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 / catalytic activity / phosphopantetheine binding / cytosol
Similarity search - Function
Condensation domain / Condensation domain / AMP-binding, conserved site / Chloramphenicol acetyltransferase-like domain superfamily / Putative AMP-binding domain signature. / 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: 2.4 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

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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)53,8552
Polymers53,7631
Non-polymers921
Water73941
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)75.759, 82.974, 86.039
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: 53762.953 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
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 41 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.51 Å3/Da / Density % sol: 51.09 %
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: CLSI / Beamline: 08ID-1 / Wavelength: 0.9537 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Sep 22, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 2.4→50 Å / Num. obs: 21197 / % possible obs: 94.2 % / Redundancy: 8.9 % / Biso Wilson estimate: 45.19 Å2 / CC1/2: 0.993 / Rpim(I) all: 0.043 / Rrim(I) all: 0.144 / Χ2: 0.666 / Net I/σ(I): 13.3
Reflection shellResolution: 2.4→2.44 Å / Mean I/σ(I) obs: 1.8 / Num. unique obs: 782 / CC1/2: 0.784 / CC star: 0.937 / Rpim(I) all: 0.288 / Rrim(I) all: 0.56 / Χ2: 0.375

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Processing

Software
NameVersionClassification
HKL-2000data collection
HKL-2000data scaling
HKL-2000data reduction
PHENIX1.19.2_4158phasing
PHENIX1.19.2_4158refinement
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.4→46.9 Å / SU ML: 0.3392 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.8144
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2652 986 4.77 %
Rwork0.2187 19686 -
obs0.2209 20672 94.77 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 58.42 Å2
Refinement stepCycle: LAST / Resolution: 2.4→46.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3454 0 6 41 3501
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00223567
X-RAY DIFFRACTIONf_angle_d0.48784842
X-RAY DIFFRACTIONf_chiral_restr0.041531
X-RAY DIFFRACTIONf_plane_restr0.0038624
X-RAY DIFFRACTIONf_dihedral_angle_d3.6493466
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.4-2.530.33611070.29312236X-RAY DIFFRACTION76.57
2.53-2.680.33011350.2882568X-RAY DIFFRACTION88.25
2.68-2.890.34541440.28282884X-RAY DIFFRACTION98.54
2.89-3.180.29941440.2622934X-RAY DIFFRACTION99.9
3.18-3.640.32541510.23372951X-RAY DIFFRACTION100
3.64-4.590.22191530.18993001X-RAY DIFFRACTION99.97
4.59-46.90.2151520.17933112X-RAY DIFFRACTION99.6
Refinement TLS params.Method: refined / Origin x: 1.91231128264 Å / Origin y: -5.02198697204 Å / Origin z: -4.93933680666 Å
111213212223313233
T0.30741322785 Å2-0.0813233283432 Å20.0185673531673 Å2-0.281160668939 Å20.0107031273193 Å2--0.295164906643 Å2
L1.60946685818 °2-0.793699096274 °2-0.637050190444 °2-1.97286826676 °2-0.221717564674 °2--1.57722667105 °2
S0.192368522414 Å °-0.0189867519027 Å °0.246956966619 Å °0.0528651372373 Å °-0.1056902419 Å °-0.0753775275389 Å °-0.413193356044 Å °0.195614267439 Å °-0.071648484902 Å °
Refinement TLS groupSelection details: all

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