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- EMDB-44495: Tyrocidine synthetase modules 1 and 2 crosslinked in the condensa... -

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Basic information

Entry
Database: EMDB / ID: EMD-44495
TitleTyrocidine synthetase modules 1 and 2 crosslinked in the condensation state, complex C
Map dataComplex C, full map
Sample
  • Complex: Complex of TycA and TycB module 1 crosslinked through the condensation domain
    • Protein or peptide: Tyrocidine synthase 1
    • Protein or peptide: Tyrocidine synthase 2
  • Ligand: (7S,10aR)-1-methyl-4-{4-[(5R)-1,1,5-trihydroxy-4,4-dimethyl-1,6,10,15-tetraoxo-2-oxa-7,11,14-triaza-1lambda~5~-phosphahexadecan-16-yl]phenyl}-3,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazin-7-yl [(5R)-1,1,5-trihydroxy-4,4-dimethyl-1,6,10-trioxo-2-oxa-7,11-diaza-1lambda~5~-phosphatridecan-13-yl]carbamate (non-preferred name)
KeywordsComplex / Crosslinked / NRPS / ISOMERASE
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
Tyrocidine synthase 2 / Tyrocidine synthase 1
Similarity search - Component
Biological speciesBrevibacillus parabrevis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.31 Å
AuthorsHeberlig GW / Burkart MD
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseOct 9, 2024-
Map releaseOct 9, 2024-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44495.png

Downloads & links

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Map

FileDownload / File: emd_44495.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComplex C, full map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.94 Å/pix.
x 280 pix.
= 261.8 Å		0.94 Å/pix.
x 280 pix.
= 261.8 Å		0.94 Å/pix.
x 280 pix.
= 261.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.935 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.42785627 - 0.7428459
Average (Standard dev.)0.0003426875 (±0.017153336)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 261.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44495_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Complex C, deepemhancer

Fileemd_44495_additional_1.map
AnnotationComplex C, deepemhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Complex C, half A

Fileemd_44495_half_map_1.map
AnnotationComplex C, half A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Complex C, half B

Fileemd_44495_half_map_2.map
AnnotationComplex C, half B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of TycA and TycB module 1 crosslinked through the condens...

EntireName: Complex of TycA and TycB module 1 crosslinked through the condensation domain
Components
  • Complex: Complex of TycA and TycB module 1 crosslinked through the condensation domain
    • Protein or peptide: Tyrocidine synthase 1
    • Protein or peptide: Tyrocidine synthase 2
  • Ligand: (7S,10aR)-1-methyl-4-{4-[(5R)-1,1,5-trihydroxy-4,4-dimethyl-1,6,10,15-tetraoxo-2-oxa-7,11,14-triaza-1lambda~5~-phosphahexadecan-16-yl]phenyl}-3,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazin-7-yl [(5R)-1,1,5-trihydroxy-4,4-dimethyl-1,6,10-trioxo-2-oxa-7,11-diaza-1lambda~5~-phosphatridecan-13-yl]carbamate (non-preferred name)

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Supramolecule #1: Complex of TycA and TycB module 1 crosslinked through the condens...

SupramoleculeName: Complex of TycA and TycB module 1 crosslinked through the condensation domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Brevibacillus parabrevis (bacteria)

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Macromolecule #1: Tyrocidine synthase 1

MacromoleculeName: Tyrocidine synthase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: phenylalanine racemase (ATP-hydrolysing)
Source (natural)Organism: Brevibacillus parabrevis (bacteria)
Molecular weightTheoretical: 124.062922 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVANQANLID NKRELEQHAL VPYAQGKSIH QLFEEQAEAF PDRVAIVFEN RRLSYQELNR KANQLARALL EKGVQTDSIV GVMMEKSIE NVIAILAVLK AGGAYVPIDI EYPRDRIQYI LQDSQTKIVL TQKSVSQLVH DVGYSGEVVV LDEEQLDARE T ANLHQPSK ...String:
MVANQANLID NKRELEQHAL VPYAQGKSIH QLFEEQAEAF PDRVAIVFEN RRLSYQELNR KANQLARALL EKGVQTDSIV GVMMEKSIE NVIAILAVLK AGGAYVPIDI EYPRDRIQYI LQDSQTKIVL TQKSVSQLVH DVGYSGEVVV LDEEQLDARE T ANLHQPSK PTDLAYVIYT SGTTGKPKGT MLEHKGIANL QSFFQNSFGV TEQDRIGLFA SMSFDASVWE MFMALLSGAS LY ILSKQTI HDFAAFEHYL SENELTIITL PPTYLTHLTP ERITSLRIMI TAGSASSAPL VNKWKDKLRY INAYGPTETS ICA TIWEAP SNQLSVQSVP IGKPIQNTHI YIVNEDLQLL PTGSEGELCI GGVGLARGYW NRPDLTAEKF VDNPFVPGEK MYRT GDLAK WLTDGTIEFL GRIDHQVKIR GHRIELGEIE SVLLAHEHIT EAVVIAREDQ HAGQYLCAYY ISQQEATPAQ LRDYA AQKL PAYMLPSYFV KLDKMPLTPN DKIDRKALPE PDLTANQSQA AYHPPRTETE SILVSIWQNV LGIEKIGIRD NFYSLG GDS IQAIQVVARL HSYQLKLETK DLLNYPTIEQ VALFVKSTTR KSDQGIIAGN VPLTPIQKWF FGKNFTNTGH WNQSSVL YR PEGFDPKVIQ SVMDKIIEHH DALRMVYQHE NGNVVQHNRG LGGQLYDFFS YNLTAQPDVQ QAIEAETQRL HSSMNLQE G PLVKVALFQT LHGDHLFLAI HHLVVDGISW RILFEDLATG YAQALAGQAI SLPEKTDSFQ SWSQWLQEYA NEADLLSEI PYWESLESQA KNVSLPKDYE VTDCKQKSVR NMRIRLHPEE TEQLLKHANQ AYQTEINDLL LAALGLAFAE WSKLAQIVIH LEGHGREDI IEQANVARTV GWFTSQYPVL LDLKQTAPLS DYIKLTKENM RKIPRKGIGY DILKHVTLPE NRGSLSFRVQ P EVTFNYLG QFDADMRTEL FTRSPYSGGN TLGADGKNNL SPESEVYTAL NITGLIEGGE LVLTFSYSSE QYREESIQQL SQ SYQKHLL AIIAHCTEKK EVERTPSDFS VKGLQMEEMD DIFELLANRL RGSRSHHHHH H

UniProtKB: Tyrocidine synthase 1

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Macromolecule #2: Tyrocidine synthase 2

MacromoleculeName: Tyrocidine synthase 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: phenylalanine racemase (ATP-hydrolysing)
Source (natural)Organism: Brevibacillus parabrevis (bacteria)
Molecular weightTheoretical: 119.775961 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGVFSKEQVQ DMYALTPMQE GMLFHALLDQ EHNSHLVQMS ISLQGDLDVG LFTDSLHVLV ERYDVFRTLF LYEKLKQPLQ VVLKQRPIP IEFYDLSACD ESEKQLRYTQ YKRADQERTF HLAKDPLMRV ALFQMSQHDY QVIWSFHHIL MDGWCFSIIF D DLLAIYLS ...String:
MGVFSKEQVQ DMYALTPMQE GMLFHALLDQ EHNSHLVQMS ISLQGDLDVG LFTDSLHVLV ERYDVFRTLF LYEKLKQPLQ VVLKQRPIP IEFYDLSACD ESEKQLRYTQ YKRADQERTF HLAKDPLMRV ALFQMSQHDY QVIWSFHHIL MDGWCFSIIF D DLLAIYLS LQNKTALSLE PVQPYSRFIN WLEKQNKQAA LNYWSDYLEA YEQKTTLPKK EAAFAKAFQP TQYRFSLNRT LT KQLGTIA SQNQVTLSTV IQTIWGVLLQ KYNAAHDVLF GSVVSGRPTD IVGIDKMVGL FINTIPFRVQ AKAGQTFSEL LQA VHKRTL QSQPYEHVPL YDIQTQSVLK QELIDHLLVI ENYPLVEALQ KKALNQQIGF TITAVEMFEP TNYDLTVMVM PKEE LAFRF DYNAALFDEQ VVQKLAGHLQ QIADCVANNS GVELCQIPLL TEAETSQLLA KRTETAADYP AATMHELFSR QAEKT PEQV AVVFADQHLT YRELDEKSNQ LARFLRKKGI GTGSLVGTLL DRSLDMIVGI LGVLKAGGAF VPIDPELPAE RIAYML THS RVPLVVTQNH LRAKVTTPTE TIDINTAVIG EESRAPIESL NQPHDLFYII YTSGTTGQPK GVMLEHRNMA NLMHFTF DQ TNIAFHEKVL QYTTCSFDVC YQEIFSTLLS GGQLYLITNE LRRHVEKLFA FIQEKQISIL SLPVSFLKFI FNEQDYAQ S FPRCVKHIIT AGEQLVVTHE LQKYLRQHRV FLHNHYGPSE THVVTTCTMD PGQAIPELPP IGKPISNTGI YILDEGLQL KPEGIVGELY ISGANVGRGY LHQPELTAEK FLDNPYQPGE RMYRTGDLAR WLPDGQLEFL GRIDHQVKIR GHRIELGEIE SRLLNHPAI KEAVVIDRAD ETGGKFLCAY VVLQKALSDE EMRAYLAQAL PEYMIPSFFV TLERIPVTPN GKTDRRALPK P EGSAKTKA DYVAPTTELE QKLVAIWEQI LGVSPIGIQD HFFTLGGHSL KAIQLISRIQ KECQADVPLR VLFEQPTIQA LA AYVELEH HHHHH

UniProtKB: Tyrocidine synthase 2

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Macromolecule #3: (7S,10aR)-1-methyl-4-{4-[(5R)-1,1,5-trihydroxy-4,4-dimethyl-1,6,1...

MacromoleculeName: (7S,10aR)-1-methyl-4-{4-[(5R)-1,1,5-trihydroxy-4,4-dimethyl-1,6,10,15-tetraoxo-2-oxa-7,11,14-triaza-1lambda~5~-phosphahexadecan-16-yl]phenyl}-3,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazin-7- ...Name: (7S,10aR)-1-methyl-4-{4-[(5R)-1,1,5-trihydroxy-4,4-dimethyl-1,6,10,15-tetraoxo-2-oxa-7,11,14-triaza-1lambda~5~-phosphahexadecan-16-yl]phenyl}-3,5,6,7,8,9,10,10a-octahydrocycloocta[d]pyridazin-7-yl [(5R)-1,1,5-trihydroxy-4,4-dimethyl-1,6,10-trioxo-2-oxa-7,11-diaza-1lambda~5~-phosphatridecan-13-yl]carbamate (non-preferred name)
type: ligand / ID: 3 / Number of copies: 1 / Formula: A1AN7
Molecular weightTheoretical: 1.01898 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.90 mg/mL
BufferpH: 7.3
Component:
ConcentrationNameFormula
50.0 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMSodium chlorideNaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 148367
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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