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- PDB-9bfg: Structure of the crosslinked PCP-E didomain of tyrocidine synthetase A -

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Basic information

Entry
Database: PDB / ID: 9bfg
TitleStructure of the crosslinked PCP-E didomain of tyrocidine synthetase A
ComponentsTyrocidine synthase 1
KeywordsBIOSYNTHETIC PROTEIN / Complex / Crosslinked / NRPS
Function / homology
Function and homology information


phenylalanine racemase (ATP-hydrolysing) / phenylalanine racemase (ATP-hydrolyzing) activity / lipid biosynthetic process / ligase activity / antibiotic biosynthetic process / ATP binding
Similarity search - Function
AMP-binding / Non-ribosomal peptide synthase / Condensation domain / Condensation domain / Amino acid adenylation domain / AMP-binding enzyme C-terminal domain / AMP-binding enzyme, C-terminal domain / Chloramphenicol acetyltransferase-like domain superfamily / AMP-binding, conserved site / Putative AMP-binding domain signature. ...AMP-binding / Non-ribosomal peptide synthase / Condensation domain / Condensation domain / Amino acid adenylation domain / AMP-binding enzyme C-terminal domain / AMP-binding enzyme, C-terminal domain / Chloramphenicol acetyltransferase-like domain superfamily / AMP-binding, conserved site / Putative AMP-binding domain signature. / AMP-dependent synthetase/ligase / AMP-binding enzyme / AMP-binding enzyme, C-terminal domain superfamily / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain
Similarity search - Domain/homology
Chem-2GH / Tyrocidine synthase 1
Similarity search - Component
Biological speciesBrevibacillus parabrevis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.18 Å
AuthorsHeberlig, G.W. / Burkart, M.D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM095970 United States
CitationJournal: Nature / Year: 2025
Title: Crosslinking intermodular condensation in non-ribosomal peptide biosynthesis.
Authors: Graham W Heberlig / James J La Clair / Michael D Burkart /
Abstract: Non-ribosomal peptide synthetases are assembly line biosynthetic pathways that are used to produce critical therapeutic drugs and are typically arranged as large multi-domain proteins called ...Non-ribosomal peptide synthetases are assembly line biosynthetic pathways that are used to produce critical therapeutic drugs and are typically arranged as large multi-domain proteins called megasynthetases. They synthesize polypeptides using peptidyl carrier proteins that shuttle each amino acid through modular loading, modification and elongation steps, and remain challenging to structurally characterize, owing in part to the inherent dynamics of their multi-domain and multi-modular architectures. Here we have developed site-selective crosslinking probes to conformationally constrain and resolve the interactions between carrier proteins and their partner enzymatic domains. We apply tetrazine click chemistry to trap the condensation of two carrier protein substrates within the active site of the condensation domain that unites the first two modules of tyrocidine biosynthesis and report the high-resolution cryo-EM structure of this complex. Together with the X-ray crystal structure of the first carrier protein crosslinked to its epimerization domain, these structures highlight captured intermodular recognition events and define the processive movement of a carrier protein from one catalytic step to the next. Characterization of these structural relationships remains central to understanding the molecular details of these unique synthetases and critically informs future synthetic biology design of these pathways.
History
DepositionApr 17, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Dec 25, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Feb 19, 2025Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tyrocidine synthase 1
B: Tyrocidine synthase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)128,78523
Polymers125,9412
Non-polymers2,84421
Water5,819323
1
A: Tyrocidine synthase 1
hetero molecules

B: Tyrocidine synthase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)128,78523
Polymers125,9412
Non-polymers2,84421
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-y+1,x-y,z1
Buried area5400 Å2
ΔGint-146 kcal/mol
Surface area42030 Å2
MethodPISA
Unit cell
Length a, b, c (Å)216.381, 216.381, 59.758
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number173
Space group name H-MP63

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Tyrocidine synthase 1 / Tyrocidine synthase I


Mass: 62970.727 Da / Num. of mol.: 2 / Mutation: E882A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Brevibacillus parabrevis (bacteria) / Gene: tycA / Production host: Escherichia coli (E. coli)
References: UniProt: P09095, phenylalanine racemase (ATP-hydrolysing)

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Non-polymers , 5 types, 344 molecules

#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID


Mass: 195.237 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-2GH / N~3~-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-N-pentyl-beta-alaninamide


Mass: 368.363 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H29N2O7P / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 323 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 3.21 Å3/Da / Density % sol: 61.64 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7.1
Details: 50 mM MES, 1.8 M ammonium sulfate, 10% v/v glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.1 / Wavelength: 0.9774 Å
DetectorType: DECTRIS PILATUS3 2M / Detector: PIXEL / Date: Dec 18, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9774 Å / Relative weight: 1
ReflectionResolution: 2.18→46.85 Å / Num. obs: 83879 / % possible obs: 100 % / Redundancy: 10.3 % / CC1/2: 0.998 / Rmerge(I) obs: 0.13 / Rpim(I) all: 0.062 / Rrim(I) all: 0.144 / Net I/σ(I): 13.6
Reflection shellResolution: 2.18→2.22 Å / Redundancy: 10.2 % / Rmerge(I) obs: 1.401 / Mean I/σ(I) obs: 1.8 / Num. unique obs: 4611 / CC1/2: 0.691 / Rpim(I) all: 0.674 / Rrim(I) all: 1.558 / % possible all: 100

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Processing

Software
NameVersionClassification
XDSdata reduction
Aimlessdata scaling
PHASERphasing
REFMAC5.8.0267refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.18→46.85 Å / Cor.coef. Fo:Fc: 0.958 / Cor.coef. Fo:Fc free: 0.936 / SU B: 5.417 / SU ML: 0.133 / Cross valid method: FREE R-VALUE / ESU R: 0.189 / ESU R Free: 0.17
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2283 4242 5.058 %
Rwork0.1882 79625 -
all0.19 --
obs-83867 99.983 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 42.664 Å2
Baniso -1Baniso -2Baniso -3
1-0.586 Å20.293 Å20 Å2
2--0.586 Å2-0 Å2
3----1.902 Å2
Refinement stepCycle: LAST / Resolution: 2.18→46.85 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8507 0 166 323 8996
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0138852
X-RAY DIFFRACTIONr_bond_other_d0.0010.0158259
X-RAY DIFFRACTIONr_angle_refined_deg1.5531.63512017
X-RAY DIFFRACTIONr_angle_other_deg1.3431.57419005
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.78651063
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.96323.411475
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.59151486
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.0731542
X-RAY DIFFRACTIONr_chiral_restr0.0710.21133
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.0210001
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022039
X-RAY DIFFRACTIONr_nbd_refined0.2050.21764
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1790.27744
X-RAY DIFFRACTIONr_nbtor_refined0.1680.24217
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.080.24339
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.140.2349
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.3850.220
X-RAY DIFFRACTIONr_nbd_other0.2360.266
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.290.27
X-RAY DIFFRACTIONr_mcbond_it3.5494.2484258
X-RAY DIFFRACTIONr_mcbond_other3.5474.2484257
X-RAY DIFFRACTIONr_mcangle_it5.0556.375319
X-RAY DIFFRACTIONr_mcangle_other5.0556.3715320
X-RAY DIFFRACTIONr_scbond_it4.6694.7534594
X-RAY DIFFRACTIONr_scbond_other4.6684.7534595
X-RAY DIFFRACTIONr_scangle_it6.9766.956698
X-RAY DIFFRACTIONr_scangle_other6.9756.9516699
X-RAY DIFFRACTIONr_lrange_it9.81281.43537552
X-RAY DIFFRACTIONr_lrange_other9.81381.46437380
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.18-2.2370.293040.2745821X-RAY DIFFRACTION99.9837
2.237-2.2980.2943080.255744X-RAY DIFFRACTION100
2.298-2.3650.2893280.2355494X-RAY DIFFRACTION100
2.365-2.4370.2612890.2325359X-RAY DIFFRACTION100
2.437-2.5170.2642590.2265254X-RAY DIFFRACTION100
2.517-2.6060.2472260.2075125X-RAY DIFFRACTION100
2.606-2.7040.2812900.2094844X-RAY DIFFRACTION100
2.704-2.8140.2722500.2064694X-RAY DIFFRACTION99.9798
2.814-2.9390.2562610.2044477X-RAY DIFFRACTION100
2.939-3.0830.2221800.1914373X-RAY DIFFRACTION100
3.083-3.250.2282400.1844100X-RAY DIFFRACTION100
3.25-3.4470.2072310.183875X-RAY DIFFRACTION100
3.447-3.6840.1922030.1753658X-RAY DIFFRACTION100
3.684-3.980.2171690.1643439X-RAY DIFFRACTION100
3.98-4.3590.1931680.1463144X-RAY DIFFRACTION100
4.359-4.8730.1791430.1422869X-RAY DIFFRACTION100
4.873-5.6270.2191240.1722552X-RAY DIFFRACTION100
5.627-6.890.2691070.2152163X-RAY DIFFRACTION100
6.89-9.7370.1741010.1641688X-RAY DIFFRACTION100
9.737-46.850.24610.22952X-RAY DIFFRACTION98.8293

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