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- EMDB-61520: Class 3 state of the GfsA KSQ-ancestralAT chimeric didomain in co... -

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Entry
Database: EMDB / ID: EMD-61520
TitleClass 3 state of the GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
Map data
Sample
  • Complex: Class 3 state of the GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
    • Protein or peptide: Polyketide synthase GfsA
    • Protein or peptide: Polyketide synthase
  • Ligand: N-[2-(acetylamino)ethyl]-N~3~-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alaninamide
Keywordsdecarboxylase / polyketide synthase / LYASE
Function / homology
Function and homology information


Lyases; Carbon-carbon lyases; Carboxy-lyases / secondary metabolite biosynthetic process / fatty acid synthase activity / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / antibiotic biosynthetic process / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / fatty acid biosynthetic process / lyase activity
Similarity search - Function
Polyketide synthase dimerisation element domain / Polyketide synthase dimerisation element domain / : / Polyketide synthase dehydratase N-terminal domain / Polyketide synthase, dehydratase domain / PKS_DH / PKS_PP_betabranch / : / Polyketide synthase dehydratase domain / : ...Polyketide synthase dimerisation element domain / Polyketide synthase dimerisation element domain / : / Polyketide synthase dehydratase N-terminal domain / Polyketide synthase, dehydratase domain / PKS_DH / PKS_PP_betabranch / : / Polyketide synthase dehydratase domain / : / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / Polyketide synthase, dehydratase domain superfamily / Polyketide synthase, C-terminal extension / 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, phosphopantetheine-binding domain / Phosphopantetheine attachment site / PKS_KR / Beta-ketoacyl synthase / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / Ketosynthase family 3 (KS3) domain profile. / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / Polyketide synthase, beta-ketoacyl synthase domain / Beta-ketoacyl synthase, N-terminal domain / Beta-ketoacyl synthase, C-terminal domain / 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
Polyketide synthase GfsA
Similarity search - Component
Biological speciesStreptomyces graminofaciens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsChisuga T / Liao Z / Adachi N / Kawasaki M / Moriya T / Senda T / Kudo F / Eguchi T / Miyanaga A
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H02911 Japan
Japan Society for the Promotion of Science (JSPS)23K19283 Japan
CitationJournal: Nat Commun / Year: 2025
Title: Ancestral sequence reconstruction as a tool for structural analysis of modular polyketide synthases.
Authors: Taichi Chisuga / Shota Takinami / Zengwei Liao / Masayuki Karasawa / Naruhiko Adachi / Masato Kawasaki / Toshio Moriya / Toshiya Senda / Tohru Terada / Fumitaka Kudo / Tadashi Eguchi / Shogo ...Authors: Taichi Chisuga / Shota Takinami / Zengwei Liao / Masayuki Karasawa / Naruhiko Adachi / Masato Kawasaki / Toshio Moriya / Toshiya Senda / Tohru Terada / Fumitaka Kudo / Tadashi Eguchi / Shogo Nakano / Sohei Ito / Akimasa Miyanaga /
Abstract: Modular polyketide synthases (PKSs) are large multi-domain enzymes critical for the biosynthesis of polyketide antibiotics. However, challenges with structural analysis limits our mechanistic ...Modular polyketide synthases (PKSs) are large multi-domain enzymes critical for the biosynthesis of polyketide antibiotics. However, challenges with structural analysis limits our mechanistic understanding of modular PKSs. In this report, we explore the potential of ancestral sequence reconstruction (ASR) for structure analysis of target proteins. As a model, we focus on the FD-891 PKS loading module composed of ketosynthase-like decarboxylase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains. We construct a KSAncAT chimeric didomain by replacing the native AT with an ancestral AT (AncAT) using ASR. After confirming that KSAncAT chimeric didomain retains similar enzymatic function to the native KSAT didomain, we successfully determine a high-resolution crystal structure of the KSAncAT chimeric didomain and cryo-EM structures of the KS-ACP complex. These cryo-EM structures, which could not be determined for the native protein, exemplify the utility of ASR to enable cryo-EM single-particle analysis. Our findings demonstrate that integrating ASR with structural analysis provides deeper mechanistic insight into modular PKSs. Furthermore, applying ASR to a partial region of the targeted multi-domain proteins could expand the potential of ASR and may serve as a valuable framework for investigating the structure and function of various multi-domain proteins.
History
DepositionSep 13, 2024-
Header (metadata) releaseAug 6, 2025-
Map releaseAug 6, 2025-
UpdateAug 13, 2025-
Current statusAug 13, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61520.png

Downloads & links

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Map

FileDownload / File: emd_61520.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
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AxesZ (Sec.)Y (Row.)X (Col.)
0.58 Å/pix.
x 512 pix.
= 296.96 Å		0.58 Å/pix.
x 512 pix.
= 296.96 Å		0.58 Å/pix.
x 512 pix.
= 296.96 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.58 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.63854414 - 0.91309834
Average (Standard dev.)-0.00028803296 (±0.015691537)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 296.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_61520_msk_1.map
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Additional map: #1

Fileemd_61520_additional_1.map
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Half map: #1

Fileemd_61520_half_map_1.map
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Half map: #2

Fileemd_61520_half_map_2.map
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Sample components

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Entire : Class 3 state of the GfsA KSQ-ancestralAT chimeric didomain in co...

EntireName: Class 3 state of the GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
Components
  • Complex: Class 3 state of the GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
    • Protein or peptide: Polyketide synthase GfsA
    • Protein or peptide: Polyketide synthase
  • Ligand: N-[2-(acetylamino)ethyl]-N~3~-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alaninamide

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Supramolecule #1: Class 3 state of the GfsA KSQ-ancestralAT chimeric didomain in co...

SupramoleculeName: Class 3 state of the GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Streptomyces graminofaciens (bacteria)

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Macromolecule #1: Polyketide synthase GfsA

MacromoleculeName: Polyketide synthase GfsA / type: protein_or_peptide / ID: 1 / Details: GfsA KSQ-ancestralAT / Number of copies: 2 / Enantiomer: LEVO
EC number: Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
Source (natural)Organism: Streptomyces graminofaciens (bacteria)
Molecular weightTheoretical: 63.183352 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: HMGRSQNSEF ETASDEPIAV IGLSCRLPKA SGPQELWQLL DDGASAVTRV PADRETPPST EEESADGEAA GARWGGFLDR VDTFDAGFF GISPREAAAM DPQQRLVLEL SWEALEGAGL VPATLRDTGL GVFVGAARDD YATLYRRREG RAVDHHAMTG L HRSLIANR ...String:
HMGRSQNSEF ETASDEPIAV IGLSCRLPKA SGPQELWQLL DDGASAVTRV PADRETPPST EEESADGEAA GARWGGFLDR VDTFDAGFF GISPREAAAM DPQQRLVLEL SWEALEGAGL VPATLRDTGL GVFVGAARDD YATLYRRREG RAVDHHAMTG L HRSLIANR ISYALGAHGP SMVVDTGCSS SLVAVHLACE SLRRGESDIA LAGGVNLNIA AESARETAAF GGLSPDGQCF TF DARANGF VRGEGGGLVV LKTLRRALAD GDLVHGVILA SAVNNDGPSD TLTTPSRRAQ ESLLTRVYRR AGVTPTEVGY VEL HGTGTK VGDPIEAAAL GAVLGTGRDT PLPVGSIKTN IGHLEGAAGI AGLIKALLQL RRRRLVPSLN FSTPNPDIPL DALN LRVQQ ESAPWATPSG GGRTLVAGVS SFGMGGTNCH VVVSAAPVPE DGETTSEAGA TGPDSGPALL PWVVSARSPQ ALRDQ AGRL AAWADSPAGR EASPVDIGWS LATSRTHFEY RAVVSGSDRD ELVASLRALA SGSPVTAAGA VDGRAEPVAL LSAVGE LFA DGYPVDWTAY FAGWPAARVE LPTYAFQRSR HWLENVPELA VS

UniProtKB: Polyketide synthase GfsA, Polyketide synthase GfsA

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Macromolecule #2: Polyketide synthase

MacromoleculeName: Polyketide synthase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces graminofaciens (bacteria)
Molecular weightTheoretical: 10.244458 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
HMPREPVTPD SDHPDPVETV RQLTAHVLGL TAAADVEMTR SFKDLGFDSL MSVELRDRLC AATGLSLATT LLYDHPSPAE TAEFVRARL TGDEA

UniProtKB: Polyketide synthase GfsA

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Macromolecule #3: N-[2-(acetylamino)ethyl]-N~3~-[(2R)-2-hydroxy-3,3-dimethyl-4-(pho...

MacromoleculeName: N-[2-(acetylamino)ethyl]-N~3~-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alaninamide
type: ligand / ID: 3 / Number of copies: 1 / Formula: 9EF
Molecular weightTheoretical: 383.335 Da
Chemical component information

ChemComp-9EF:
N-[2-(acetylamino)ethyl]-N~3~-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alaninamide

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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

Concentration4.0 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
5.0 mMHEPES-NaOH4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
50.0 mMNaClsodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 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) / Average electron dose: 50.0 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 105895
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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