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- EMDB-41307: KS-AT core of 6-deoxyerythronolide B synthase (DEBS) Module 3 cro... -

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Basic information

Entry
Database: EMDB / ID: EMD-41307
TitleKS-AT core of 6-deoxyerythronolide B synthase (DEBS) Module 3 crosslinked with its elongation ACP partner
Map data
Sample
  • Complex: KS-AT core of DEBS Module 3 crosslinked with its elongation ACP partner
    • Protein or peptide: EryAII
Keywordspolyketide synthase / antibody / BIOSYNTHETIC PROTEIN
Function / homology
Function and homology information


macrolide biosynthetic process / DIM/DIP cell wall layer assembly / fatty acid synthase activity / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process / oxidoreductase activity / plasma membrane / cytoplasm
Similarity search - Function
Erythronolide synthase, docking / Erythronolide synthase, docking domain superfamily / Erythronolide synthase, docking / Polyketide synthase extender module SpnB, Rossmann fold domain / Polyketide synthase, docking domain / Erythronolide synthase docking domain / Zinc-binding dehydrogenase / : / Polyketide synthase dehydratase N-terminal domain / Polyketide synthase, dehydratase domain ...Erythronolide synthase, docking / Erythronolide synthase, docking domain superfamily / Erythronolide synthase, docking / Polyketide synthase extender module SpnB, Rossmann fold domain / Polyketide synthase, docking domain / Erythronolide synthase docking domain / Zinc-binding dehydrogenase / : / Polyketide synthase dehydratase N-terminal domain / Polyketide synthase, dehydratase domain / PKS_DH / : / Polyketide synthase dehydratase domain / Polyketide synthase, dehydratase domain superfamily / : / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / Polyketide synthase, C-terminal extension / PKS_PP_betabranch / Ketoacyl-synthetase C-terminal extension / Polyketide synthase, ketoreductase domain / KR domain / Malonyl-CoA ACP transacylase, ACP-binding / : / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase domain superfamily / Acyl transferase/acyl hydrolase/lysophospholipase / Polyketide synthase, enoylreductase domain / Enoylreductase / Alcohol dehydrogenase, N-terminal / Alcohol dehydrogenase GroES-like domain / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / PKS_KR / Ketosynthase family 3 (KS3) domain profile. / Polyketide synthase, beta-ketoacyl synthase domain / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / Beta-ketoacyl synthase, N-terminal domain / Beta-ketoacyl synthase, C-terminal domain / GroES-like superfamily / Thiolase-like / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Biological speciesSaccharopolyspora erythraea (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsCogan DP / Soohoo AM / Chen M / Brodsky KL / Liu Y / Khosla C
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM141799 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136039 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM129541 United States
National Science Foundation (NSF, United States)DGE-1656518 United States
CitationJournal: Nat Chem Biol / Year: 2024
Title: Structural basis for intermodular communication in assembly-line polyketide biosynthesis.
Authors: Dillon P Cogan / Alexander M Soohoo / Muyuan Chen / Yan Liu / Krystal L Brodsky / Chaitan Khosla /
Abstract: Assembly-line polyketide synthases (PKSs) are modular multi-enzyme systems with considerable potential for genetic reprogramming. Understanding how they selectively transport biosynthetic ...Assembly-line polyketide synthases (PKSs) are modular multi-enzyme systems with considerable potential for genetic reprogramming. Understanding how they selectively transport biosynthetic intermediates along a defined sequence of active sites could be harnessed to rationally alter PKS product structures. To investigate functional interactions between PKS catalytic and substrate acyl carrier protein (ACP) domains, we employed a bifunctional reagent to crosslink transient domain-domain interfaces of a prototypical assembly line, the 6-deoxyerythronolide B synthase, and resolved their structures by single-particle cryogenic electron microscopy (cryo-EM). Together with statistical per-particle image analysis of cryo-EM data, we uncovered interactions between ketosynthase (KS) and ACP domains that discriminate between intra-modular and inter-modular communication while reinforcing the relevance of conformational asymmetry during the catalytic cycle. Our findings provide a foundation for the structure-based design of hybrid PKSs comprising biosynthetic modules from different naturally occurring assembly lines.
History
DepositionJul 23, 2023-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_41307.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 448 pix.
= 423.808 Å
0.95 Å/pix.
x 448 pix.
= 423.808 Å
0.95 Å/pix.
x 448 pix.
= 423.808 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.946 Å
Density
Contour LevelBy AUTHOR: 0.339
Minimum - Maximum-0.46599337 - 0.9122427
Average (Standard dev.)0.0002786378 (±0.02250235)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 423.80798 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_41307_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_41307_half_map_2.map
Projections & Slices
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Sample components

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Entire : KS-AT core of DEBS Module 3 crosslinked with its elongation ACP p...

EntireName: KS-AT core of DEBS Module 3 crosslinked with its elongation ACP partner
Components
  • Complex: KS-AT core of DEBS Module 3 crosslinked with its elongation ACP partner
    • Protein or peptide: EryAII

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Supramolecule #1: KS-AT core of DEBS Module 3 crosslinked with its elongation ACP p...

SupramoleculeName: KS-AT core of DEBS Module 3 crosslinked with its elongation ACP partner
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharopolyspora erythraea (bacteria)
Molecular weightTheoretical: 210 KDa

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Macromolecule #1: EryAII

MacromoleculeName: EryAII / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharopolyspora erythraea (bacteria)
Molecular weightTheoretical: 99.76575 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MVTDSEKVAE YLRRATLDLR AARQRIRELE SDPIAIVSMA CRLPGGVNTP QRLWELLREG GETLSGFPTD RGWDLARLHH PDPDNPGTS YVDKGGFLDD AAGFDAEFFG VSPREAAAMD PQQRLLLETS WELVENAGID PHSLRGTATG VFLGVAKFGY G EDTAAAED ...String:
MVTDSEKVAE YLRRATLDLR AARQRIRELE SDPIAIVSMA CRLPGGVNTP QRLWELLREG GETLSGFPTD RGWDLARLHH PDPDNPGTS YVDKGGFLDD AAGFDAEFFG VSPREAAAMD PQQRLLLETS WELVENAGID PHSLRGTATG VFLGVAKFGY G EDTAAAED VEGYSVTGVA PAVASGRISY TMGLEGPSIS VDTACSSSLV ALHLAVESLR KGESSMAVVG GAAVMATPGV FV DFSRQRA LAADGRSKAF GAGADGFGFS EGVTLVLLER LSEARRNGHE VLAVVRGSAL NQDGASNGLS APSGPAQRRV IRQ ALESCG LEPGDVDAVE AHGTGTALGD PIEANALLDT YGRDRDADRP LWLGSVKSNI GHTQAAAGVT GLLKVVLALR NGEL PATLH VEEPTPHVDW SSGGVALLAG NQPWRRGERT RRAAVSAFGI SGTNAHVIVE EAPEREHRET TAHDGRPVPL VVSAR STAA LRAQAAQIAE LLERPDADLA GVGLGLATTR ARHEHRAAVV ASTREEAVRG LREIAAGAAT ADAVVEGVTE VDGRNV VFL FPGQGSQWAG MGAELLSSSP VFAGKIRACD ESMAPMQDWK VSDVLRQAPG APGLDRVDVV QPVLFAVMVS LAELWRS YG VEPAAVVGHS QGEIAAAHVA GALTLEDAAK LVVGRSRLMR SLSGEGGMAA VALGEAAVRE RLRPWQDRLS VAAVNGPR S VVVSGEPGAL RAFSEDCAAE GIRVRDIDVD YASHSPQIER VREELLETTG DIAPRPARVT FHSTVESRSM DGTELDARY WYRNLRETVR FADAVTRLAE SGYDAFIEVS PHPVVVQAVE EAVEEADGAE DAVVVGSLHR DGGDLSAFLR SMATAHVSGV DIRWDVALP GAAPFALPTY PFQRKRYWLQ PAAPAAASDE LAYRSSSVDK LAAALEHHHH HH

UniProtKB: EryAII

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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

Concentration8 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
100.0 mMC6H8O7citric acid
100.0 mMNaClsodium chloride
10.0 mMC8H18N2O4SHEPES
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 11 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: 30 s glow, 10 s hold, 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 4601 / Average exposure time: 6.45 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 150.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 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

Particle selectionNumber selected: 199350
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 199350
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: OTHER
Final 3D classificationNumber classes: 8 / Avg.num./class: 40000

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8tjp:
KS-AT core of 6-deoxyerythronolide B synthase (DEBS) Module 3 crosslinked with its elongation ACP partner

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