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- EMDB-27094: AT from first module of the pikromycin synthase -

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Basic information

Entry
Database: EMDB / ID: EMD-27094
TitleAT from first module of the pikromycin synthase
Map dataLocal refinement map for one PikAT1 monomer from the engineered synthase Pik127.
Sample
  • Complex: Pik127
    • Protein or peptide: Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2
Keywordsacyltransferase / methylmalonyl-specific / polyketide synthase / BIOSYNTHETIC PROTEIN
Function / homology
Function and homology information


10-deoxymethynolide synthase / narbonolide synthase / macrolide biosynthetic process / DIM/DIP cell wall layer assembly / fatty acid synthase activity / acyltransferase activity, transferring groups other than amino-acyl groups / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process / plasma membrane / cytoplasm
Similarity search - Function
Polyketide synthase extender module SpnB, Rossmann fold domain / RhiE-like, KS-MAT linker domain / : / Polyketide synthase dehydratase N-terminal domain / Polyketide synthase, dehydratase domain / PKS_DH / : / Polyketide synthase dehydratase domain / Polyketide synthase, dehydratase domain superfamily / : ...Polyketide synthase extender module SpnB, Rossmann fold domain / RhiE-like, KS-MAT linker domain / : / 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, 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 / 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
Narbonolide/10-deoxymethynolide synthase PikA1, modules 1 and 2
Similarity search - Component
Biological speciesStreptomyces venezuelae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 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 24, 2022-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27094.png

Downloads & links

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Map

FileDownload / File: emd_27094.map.gz / Format: CCP4 / Size: 1.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refinement map for one PikAT1 monomer from the engineered synthase Pik127.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.81 Å/pix.
x 63 pix.
= 51.03 Å		0.81 Å/pix.
x 77 pix.
= 62.37 Å		0.81 Å/pix.
x 60 pix.
= 48.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.81 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.3
Minimum - Maximum-6.9035516 - 14.423334000000001
Average (Standard dev.)0.000000000087096 (±0.935854)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin37135120
Dimensions776063
Spacing637760
CellA: 51.03 Å / B: 62.37 Å / C: 48.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: First half map

Fileemd_27094_half_map_1.map
AnnotationFirst half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Second half map

Fileemd_27094_half_map_2.map
AnnotationSecond 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

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Supramolecule #1: Pik127

SupramoleculeName: Pik127 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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
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 / Details: First AT domain of pikromycin synthase / Number of copies: 1 / Enantiomer: LEVO / EC number: 10-deoxymethynolide synthase
Source (natural)Organism: Streptomyces venezuelae (bacteria)
Molecular weightTheoretical: 33.011234 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GVGRVAFVFP GQGTQWAGMG AELLDSSAVF AAAMAECEAA LSPYVDWSLE AVVRQAPGAP TLERVDVVQP VTFAVMVSLA RVWQHHGVT PQAVVGHSQG EIAAAYVAGA LSLDDAARVV TLRSKSIAAH LAGKGGMLSL ALSEDAVLER LAGFDGLSVA A VNGPTATV ...String:
GVGRVAFVFP GQGTQWAGMG AELLDSSAVF AAAMAECEAA LSPYVDWSLE AVVRQAPGAP TLERVDVVQP VTFAVMVSLA RVWQHHGVT PQAVVGHSQG EIAAAYVAGA LSLDDAARVV TLRSKSIAAH LAGKGGMLSL ALSEDAVLER LAGFDGLSVA A VNGPTATV VSGDPVQIEE LARACEADGV RARVIPVDYA SHSRQVEIIE SELAEVLAGL SPQAPRVPFF STLEGAWITE PV LDGGYWY RNLRHRVGFA PAVETLATDE GFTHFVEVSA HPVLTMALPG TVTGLATLRR DNGGQDRLVA SLAEAWAN

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

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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 mMC21H30N7O17P3NADPH

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 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
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
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
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 FIELD / 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
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.
Startup modelType of model: NONE / Details: Ab initio generated in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0)
Details: Maps were density modified using Phenix to a calculated resolution of 2.77 Angstroms.
Number images used: 184737
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.2.0) / Details: Ab initio generated in cryoSPARC
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.2.0)
Final 3D classificationNumber classes: 4 / Avg.num./class: 78019 / Software - Name: cryoSPARC (ver. 3.2.0)
Details: The selected class contained 184,737 particles. These were C2 symmetry expanded, and local refinement using a mask around the AT was performed.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2qo3


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Phenix
Output model

PDB-8czc:
AT from first module of the pikromycin synthase

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