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- PDB-9bfe: Tyrocidine synthetase modules 1 and 2 crosslinked in the condensa... -

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Basic information

Entry
Database: PDB / ID: 9bfe
TitleTyrocidine synthetase modules 1 and 2 crosslinked in the condensation state, complex B
Components
  • Tyrocidine synthase 1
  • Tyrocidine synthase 2
KeywordsISOMERASE / Complex / Crosslinked / NRPS
Function / homology
Function and homology information


phenylalanine racemase (ATP-hydrolysing) / phenylalanine racemase (ATP-hydrolyzing) activity / amino acid activation for nonribosomal peptide biosynthetic process / secondary metabolite biosynthetic process / ligase activity / phosphopantetheine binding / antibiotic biosynthetic process / ATP binding / cytoplasm
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 / AMP-binding, conserved site / Chloramphenicol acetyltransferase-like domain superfamily / 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 / AMP-binding, conserved site / Chloramphenicol acetyltransferase-like domain superfamily / Putative AMP-binding domain signature. / AMP-dependent synthetase/ligase / AMP-binding enzyme / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / 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
: / ADENOSINE MONOPHOSPHATE / Tyrocidine synthase 2 / Tyrocidine synthase 1
Similarity search - Component
Biological speciesBrevibacillus parabrevis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 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 1.1Oct 30, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.2Dec 18, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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 / _em_admin.last_update
Revision 1.3Dec 25, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.4Feb 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _em_admin.last_update

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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 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)245,1344
Polymers243,8392
Non-polymers1,2952
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Tyrocidine synthase 1 / Tyrocidine synthase I


Mass: 124062.922 Da / Num. of mol.: 1
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)
#2: Protein Tyrocidine synthase 2 / Tyrocidine synthase II


Mass: 119775.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Brevibacillus parabrevis (bacteria) / Gene: tycB / Production host: Escherichia coli (E. coli)
References: UniProt: O30408, phenylalanine racemase (ATP-hydrolysing)
#3: Chemical ChemComp-A1AN1 / (7S,10aR)-1-methyl-4-{4-[(5R)-1,1,5-trihydroxy-4,4-dimethyl-1,6,11-trioxo-2-oxa-7,10-diaza-1lambda~5~-phosphadodecan-12-yl]phenyl}-3,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazin-7-yl [2-({N-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl]carbamate


Mass: 947.902 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C39H63N7O16P2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O7P / Comment: AMP*YM
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of TycA and TycB module 1 crosslinked through the condensation domain, complex B
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Brevibacillus parabrevis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid1
2150 mMSodium chlorideNaCl1
38 mMAdenosine triphosphate1
410 mMMagnesium chlorideMgCl21
52 mML-Proline1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 55 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris X

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4cryoSPARCCTF correction
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 214241 / Symmetry type: POINT
RefinementCross valid method: NONE

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