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- PDB-8z0s: Cryo-EM structure of trimer HtmB2-CT -

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Basic information

Entry
Database: PDB / ID: 8z0s
TitleCryo-EM structure of trimer HtmB2-CT
Componentsspecial condensation domain in NRPS
KeywordsBIOSYNTHETIC PROTEIN / Dimer of special condensation domain of NRPS
Biological speciesStreptomyces (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsSun, Y.H. / Zhang, Z.Y. / Mei, Q.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31800052 China
CitationJournal: Angew Chem Int Ed Engl / Year: 2025
Title: Formation of the Diketopiperazine Moiety by a Distinct Condensation-Like Domain in Hangtaimycin Biosynthesis.
Authors: Qing Mei / Shijuan Wu / Minghe Luo / Shunjia Ji / Jiayi Guo / Chuan Dong / Guo Sun / Jian Wang / Zixin Deng / Yi-Lei Zhao / Zhengyu Zhang / Yuhui Sun /
Abstract: Non-ribosomal peptide synthetases (NRPSs) are key enzymes in pharmaceutical synthesis, with condensation (C) domains catalyzing amide bond formation between aminoacyl substrates. However, recent ...Non-ribosomal peptide synthetases (NRPSs) are key enzymes in pharmaceutical synthesis, with condensation (C) domains catalyzing amide bond formation between aminoacyl substrates. However, recent research has elucidated that the catalytic capabilities of C domains extend beyond the traditional formation of peptide bonds. In this study, we elucidate the cyclization mechanism of the NRPS-derived natural products hangtaimycin (HTM), characterized by the formation of a 2,5-diketopiperazine (DKP) moiety which involves an intramolecular vinylamide-mediated nucleophilic attack instead of an N-terminal amino group. This cyclization is catalyzed by a terminal condensation-like (C) domain within the NRPS enzyme HtmB2. We investigated the evolutionary specificity of the HtmB2-C within Streptomyces spectabilis CCTCC M2017417. Employing a multidisciplinary analytical approach, we have delineated the molecular underpinnings of DKP formation within the HTM biosynthesis. This process is facilitated by residue R2776, which modulates the formation of reactive species and stabilizes the amidate through electrostatic interactions. Besides, we found a positive correlation between the alkaline strength of the residue at position 2776 and the activity of HtmB2-C. Our study elucidates the formation mechanism of DKPs in NRPS-derived natural products, thereby bridging a critical gap in the structural and mechanistic understanding of this field.
History
DepositionApr 10, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release
Revision 1.1May 28, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Jun 18, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: special condensation domain in NRPS
C: special condensation domain in NRPS
D: special condensation domain in NRPS


Theoretical massNumber of molelcules
Total (without water)185,4683
Polymers185,4683
Non-polymers00
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 special condensation domain in NRPS


Mass: 61822.629 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces (bacteria) / Production host: Escherichia coli (E. coli)
Has protein modificationN

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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: trimer of HtmB2-CT / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Streptomyces (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 8 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 471903 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310580
ELECTRON MICROSCOPYf_angle_d0.50614421
ELECTRON MICROSCOPYf_dihedral_angle_d4.2421488
ELECTRON MICROSCOPYf_chiral_restr0.041605
ELECTRON MICROSCOPYf_plane_restr0.0051906

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