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- EMDB-60989: Class 2 state of the GfsA KSQ-ancestralAT chimeric didomain in co... -

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Entry
Database: EMDB / ID: EMD-60989
TitleClass 2 state of the GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
Map data
Sample
  • Complex: GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
Keywordsantibiotic biosynthesis / ANTIBIOTIC
Biological speciesStreptomyces graminofaciens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.73 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
DepositionJul 30, 2024-
Header (metadata) releaseAug 6, 2025-
Map releaseAug 6, 2025-
UpdateAug 27, 2025-
Current statusAug 27, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60989.png

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Map

FileDownload / File: emd_60989.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.58 Å
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Contour LevelBy AUTHOR: 0.035
Minimum - Maximum-0.17048818 - 0.3096329
Average (Standard dev.)-0.00029314114 (±0.0056168903)
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_60989_msk_1.map
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Additional map: #1

Fileemd_60989_additional_1.map
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Half map: #1

Fileemd_60989_half_map_1.map
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Half map: #2

Fileemd_60989_half_map_2.map
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Sample components

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Entire : GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA A...

EntireName: GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
Components
  • Complex: GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain

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Supramolecule #1: GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA A...

SupramoleculeName: GfsA KSQ-ancestralAT chimeric didomain in complex with the GfsA ACP domain
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Streptomyces graminofaciens (bacteria)
Molecular weightTheoretical: 108 kDa/nm

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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: NONE
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 98786
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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