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- EMDB-26839: KSQ+AT from first module of the pikromycin synthase -

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Basic information

Entry
Database: EMDB / ID: EMD-26839
TitleKSQ+AT from first module of the pikromycin synthase
Map dataPrimary map. Density fades for distal AT domains. Use AT_newest.ccp4 for these (related through twofold of PikKSAT1_dimer.cif).
Sample
  • Complex: Pik127
    • Protein or peptide: Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2
  • Ligand: water
Function / homology
Function and homology information


10-deoxymethynolide synthase / narbonolide synthase / macrolide biosynthetic process / acyltransferase activity, transferring groups other than amino-acyl groups / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process
Similarity search - Function
Polyketide synthase dehydratase N-terminal domain / PKS_DH / Polyketide synthase, dehydratase domain / Polyketide synthase, dehydratase domain superfamily / Polyketide synthase, ketoreductase domain / KR domain / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Malonyl-CoA ACP transacylase, ACP-binding / Acyl transferase domain superfamily ...Polyketide synthase dehydratase N-terminal domain / PKS_DH / Polyketide synthase, dehydratase domain / Polyketide synthase, dehydratase domain superfamily / Polyketide synthase, ketoreductase domain / KR domain / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Malonyl-CoA ACP transacylase, ACP-binding / Acyl transferase domain superfamily / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase/acyl hydrolase/lysophospholipase / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / 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 / Phosphopantetheine attachment site / Thiolase-like / 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
Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2
Similarity search - Component
Biological speciesStreptomyces venezuelae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsKeatinge-Clay AT / Dickinson MS / Miyazawa T / McCool RS
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM106112 United States
Welch FoundationF-1712 United States
CitationJournal: Structure / Year: 2022
Title: Priming enzymes from the pikromycin synthase reveal how assembly-line ketosynthases catalyze carbon-carbon chemistry.
Authors: Miles S Dickinson / Takeshi Miyazawa / Ryan S McCool / Adrian T Keatinge-Clay /
Abstract: The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KS, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups ...The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KS, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups to growing polyketide chains, it performs an extension-decoupled decarboxylation of these groups to generate primer units. When Pik127, a model triketide synthase constructed from modules of the pikromycin synthase, was studied by cryoelectron microscopy (cryo-EM), the dimeric didomain comprised of KS and the neighboring methylmalonyl-selective acyltransferase (AT) dominated the class averages and yielded structures at 2.5- and 2.8-Å resolution, respectively. Comparisons with ketosynthases complexed with their substrates revealed the conformation of the (2S)-methylmalonyl-S-phosphopantetheinyl portion of KS and KS substrates prior to decarboxylation. Point mutants of Pik127 probed the roles of residues in the KS active site, while an AT-swapped version of Pik127 demonstrated that KS can also decarboxylate malonyl groups. Mechanisms for how KS and KS domains catalyze carbon-carbon chemistry are proposed.
History
DepositionMay 3, 2022-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateSep 14, 2022-
Current statusSep 14, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26839.png

Downloads & links

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Map

FileDownload / File: emd_26839.map.gz / Format: CCP4 / Size: 5.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map. Density fades for distal AT domains. Use AT_newest.ccp4 for these (related through twofold of PikKSAT1_dimer.cif).
Voxel sizeX=Y=Z: 0.81 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-3.1150494 - 6.100517
Average (Standard dev.)1.140399e-12 (±0.51408863)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin6310698
Dimensions17575105
Spacing10517575
CellA: 85.05 Å / B: 141.75 Å / C: 60.75 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Map for AT (Chain B) obtained through local...

Fileemd_26839_additional_1.map
AnnotationMap for AT (Chain B) obtained through local refinement. Apply twofold from PikKSQAT1.cif to obtain map for AT (Chain A).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: second half map

Fileemd_26839_half_map_1.map
Annotationsecond half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: first half map

Fileemd_26839_half_map_2.map
Annotationfirst half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Pik127

EntireName: Pik127
Components
  • Complex: Pik127
    • Protein or peptide: Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2
  • Ligand: water

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Supramolecule #1: Pik127

SupramoleculeName: Pik127 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Polyketide synthase engineered from the first, second, and seventh modules of the pikromycin synthase as described in Miyazawa et al., 2021, Chem. Commun. 57:8762-8765
Source (natural)Organism: Streptomyces venezuelae (bacteria) / Strain: 15439
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: K207-3
Molecular weightTheoretical: 410 KDa

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Macromolecule #1: Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2

MacromoleculeName: Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: 10-deoxymethynolide synthase
Source (natural)Organism: Streptomyces venezuelae (bacteria)
Molecular weightTheoretical: 91.797898 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EPVAVVGISC RVPGARDPRE FWELLAAGGQ AVTDVPADRW NAGDFYDPDR SAPGRSNSRW GGFIEDVDRF DAAFFGISPR EAAEMDPQQ RLALELGWEA LERAGIDPSS LTGTRTGVFA GAIWDDYATL KHRQGGAAIT PHTVTGLHRG IIANRLSYTL G LRGPSMVV ...String:
EPVAVVGISC RVPGARDPRE FWELLAAGGQ AVTDVPADRW NAGDFYDPDR SAPGRSNSRW GGFIEDVDRF DAAFFGISPR EAAEMDPQQ RLALELGWEA LERAGIDPSS LTGTRTGVFA GAIWDDYATL KHRQGGAAIT PHTVTGLHRG IIANRLSYTL G LRGPSMVV DSGQSSSLVA VHLACESLRR GESELALAGG VSLNLVPDSI IGASKFGGLS PDGRAYTFDA RANGYVRGEG GG FVVLKRL SRAVADGDPV LAVIRGSAVN NGGAAQGMTT PDAQAQEAVL REAHERAGTA PADVRYVELH GTGTPVGDPI EAA ALGAAL GTGRPAGQPL LVGSVKTNIG HLEGAAGIAG LIKAVLAVRG RALPASLNYE TPNPAIPFEE LNLRVNTEYL PWEP EHDGQ RMVVGVSSFG MGGTNAHVVL EEAPGVVEGA SVVESTVGGS AVGGGVVPWV VSAKSAAALD AQIERLAAFA SRDRT DGVD AGAVDAGAVD AGAVARVLAG GRAQFEHRAV VVGSGPDDLA AALAAPEGLV RGVASGVGRV AFVFPGQGTQ WAGMGA ELL DSSAVFAAAM AECEAALSPY VDWSLEAVVR QAPGAPTLER VDVVQPVTFA VMVSLARVWQ HHGVTPQAVV GHSQGEI AA AYVAGALSLD DAARVVTLRS KSIAAHLAGK GGMLSLALSE DAVLERLAGF DGLSVAAVNG PTATVVSGDP VQIEELAR A CEADGVRARV IPVDYASHSR QVEIIESELA EVLAGLSPQA PRVPFFSTLE GAWITEPVLD GGYWYRNLRH RVGFAPAVE TLATDEGFTH FVEVSAHPVL TMALPGTVTG LATLRRDNGG QDRLVASLAE AWANGLAVDW SPLLPSATGH HSDLPTYAFQ TERHWL

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 55 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration1.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
200.0 mMK3PO4potassium phosphate
0.5 mMC9H15O6PTCEP
3.0 mMC25H40N7O19P3Smethylmalonyl-CoA
1.0 mMC21H30N7O17P3NADPHNicotinamide adenine dinucleotide phosphate

Details: Buffer was made fresh because TCEP rapidly degrades in phosphate
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Blotted for 3 seconds with a force of "1" before plunging..
DetailsThis sample was monodisperse. It was incubated with its substrates, methylmalonyl-CoA and NADPH, for 30 min. at 25 C before freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 61728 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsCollected 4284 movies at 0 degrees tilt and 2912 movies at -30 degrees tilt
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 7196 / Average exposure time: 5.0 sec. / Average electron dose: 70.0 e/Å2 / Details: 20 frames per second
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6761206
Details: From blob picker, 6,761,206 particles were selected. Then, from template picker, 2,350,020 particles were selected.
CTF correctionSoftware - Name: cryoSPARC (ver. 3.2.0)
Details: CTF correction performed by cryosparc during reconstruction
Startup modelType of model: NONE / Details: Ab initio generated in cryoSPARC
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.2.0) / Details: Ab initio generated in cryoSPARC
Final 3D classificationNumber classes: 4 / Avg.num./class: 78019 / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.2.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Details: Density modification in Phenix / Number images used: 184737
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2qo3

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Phenix
Output model

PDB-7uwr:
KSQ+AT from first module of the pikromycin synthase

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