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- EMDB-4266: ACP2 crosslinked to the KS of the loading/condensing region of th... -

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Basic information

Entry
Database: EMDB / ID: EMD-4266
TitleACP2 crosslinked to the KS of the loading/condensing region of the CTB1 PKS
Map data
Sample
  • Complex: CTB1-SAT0-KS-MAT0=ACP2
    • Protein or peptide: Polyketide synthase
    • Protein or peptide: Polyketide synthase
Function / homology
Function and homology information


secondary metabolite biosynthetic process / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / fatty acid biosynthetic process
Similarity search - Function
Polyketide product template domain / Starter unit:ACP transacylase / Starter unit:ACP transacylase in aflatoxin biosynthesis / Polyketide synthase, thioesterase domain / Thioesterase / CurL-like, PKS C-terminal / Thioesterase / Thioesterase domain / Polyketide synthase, dehydratase domain superfamily / : ...Polyketide product template domain / Starter unit:ACP transacylase / Starter unit:ACP transacylase in aflatoxin biosynthesis / Polyketide synthase, thioesterase domain / Thioesterase / CurL-like, PKS C-terminal / Thioesterase / Thioesterase domain / Polyketide synthase, dehydratase domain superfamily / : / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / 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. / 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 / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Non-reducing polyketide synthase CTB1
Similarity search - Component
Biological speciesCercospora nicotianae (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.1 Å
AuthorsHerbst DA / Huitt-Roehl CR / Jakob RP / Townsend CA / Maier T
Funding support Switzerland, United States, 5 items
OrganizationGrant numberCountry
Swiss National Science Foundation159696 Switzerland
Swiss National Science Foundation164074 Switzerland
Swiss National Science Foundation145023 Switzerland
National Institutes of HealthES001670 United States
Swiss National Science Foundation138262 Switzerland
CitationJournal: Nat Chem Biol / Year: 2018
Title: The structural organization of substrate loading in iterative polyketide synthases.
Authors: Dominik A Herbst / Callie R Huitt-Roehl / Roman P Jakob / Jacob M Kravetz / Philip A Storm / Jamie R Alley / Craig A Townsend / Timm Maier /
Abstract: Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an ...Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/-condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.
History
DepositionJan 18, 2018-
Header (metadata) releaseFeb 14, 2018-
Map releaseMar 21, 2018-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.031
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.031
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6fik
  • Surface level: 0.031
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4266.png

Downloads & links

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Map

FileDownload / File: emd_4266.map.gz / Format: CCP4 / Size: 35.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 210 pix.
= 278.46 Å		1.33 Å/pix.
x 210 pix.
= 278.46 Å		1.33 Å/pix.
x 210 pix.
= 278.46 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.326 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.031 / Movie #1: 0.031
Minimum - Maximum-0.066375345 - 0.1258531
Average (Standard dev.)0.00004180526 (±0.0051505407)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 278.46 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3261.3261.326
M x/y/z210210210
origin x/y/z0.0000.0000.000
length x/y/z278.460278.460278.460
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS210210210
D min/max/mean-0.0660.1260.000

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Supplemental data

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Sample components

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Entire : CTB1-SAT0-KS-MAT0=ACP2

EntireName: CTB1-SAT0-KS-MAT0=ACP2
Components
  • Complex: CTB1-SAT0-KS-MAT0=ACP2
    • Protein or peptide: Polyketide synthase
    • Protein or peptide: Polyketide synthase

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Supramolecule #1: CTB1-SAT0-KS-MAT0=ACP2

SupramoleculeName: CTB1-SAT0-KS-MAT0=ACP2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Cercospora nicotianae (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Polyketide synthase

MacromoleculeName: Polyketide synthase / type: protein_or_peptide / ID: 1 / Details: C553 in chain A is crosslinked to S1816 in chain C / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Cercospora nicotianae (fungus)
Molecular weightTheoretical: 140.255 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEDGAQMRVV AFGDQTYDCS EAVSQLLRVR DDAIVVDFLE RAPAVLKAEL ARLSSEQQEE TPRFATLAEL VPRYRAGTLN PAVSQALTC IAQLGLFIRQ HSSGQEAYPT AHDSCITGVA TGALTAVAVG SASSVTALVP LALHTVAVAV RLGARAWEIG S CLADARRG ...String:
MEDGAQMRVV AFGDQTYDCS EAVSQLLRVR DDAIVVDFLE RAPAVLKAEL ARLSSEQQEE TPRFATLAEL VPRYRAGTLN PAVSQALTC IAQLGLFIRQ HSSGQEAYPT AHDSCITGVA TGALTAVAVG SASSVTALVP LALHTVAVAV RLGARAWEIG S CLADARRG ANGRYASWTS AVGGISPQDL QDRISAYTAE QALASVSVPY LSAAVGPGQS SVSAAPVILD AFLSTLLRPL TT TRLPITA PYHAPHLFTA KDVQHVTDCL PPSEAWPTVR IPIISFSRDE AVSRGASFPA AMSEAVRDCL IRPIALDRMA VSI ANHARD LGKDSVLPSP IALSFSDKLG PQVNSHLPGA KAPTPELTSK SIPSAIGAEQ QPMAKSPIAI LAASGRFPQS SSMD QFWDV LINGVDTHEL VPPTRWNAAT HVSEDPKAKN VSGTGFGCWL HEAGEFDAAY FNMSPREAPQ VDPAQRLALL TATEA LEQA GVVPNRTSST QKNRVGVWYG ATSNDWMETN SAQNVDTYFI PGGNRAFIPG RVNYFHKFSG PSYTIDTACS SSLAAL HMA CNALWRGEVD TAIVGGTNVL TNPDMTAGLD AGHFLSRSGN CKTFDDEADG YCRGEAVVTL ILKRLPDAQA DKDPIQA SI LGIATNHSAE AASITRPHAG AQQDLFQQVL TETGLTANDI SVCEMHGTGT QAGDSGETTS VVETLAPLNR SGSAVRTT P LYIGAVKSNV GHAESAAGVS SLAKILLMLK HSKIPPHVGI KTKLNHRLPD LAARNTHIAR SEVPWPRPKN GKRRVLLNN FSAAGGNTCL VLEDAPEPED SQEVDPREHH IVALSAKTPD SMVNNLTNMI TWIDKHSGDS LATLPQLSYT TTARRVHHRH RAVATGTDL LQIRSSLQEQ LDRRVSGERS IPHPPNGPSF VLAFTGQGSA FAGMGVDLYK RFASFRSDIA RYDQICEGMS L PSIKAMFE DEKVFSTASP TLQQLTHVCF QMALYRLWKS LGVQAKAVVG HALGEYAALY AAGVLSQSDT LYLVGRRAQL ME KHLSQGT HAMLAVRAKE EAIVAAIDGP PGEAYEFSCR NGEQRNVLGG TVAQIQAAKA ALEAKKIRCQ YLDTPMAFHT GQV DPILPE LLQVAAACSI QDPQIPVISP AYGKVIRSAK DFQPEYFTHH CRSSVNMVDA LQSAVEEGLL DKNVIGLEIG PGPV VTQFV KEAVGTTMQT FASINKDKDT WQLMTQALAK FYLAGASVEW SRYHEDFPGA QKVLELPAYG WALKNYWLQY VNDWS LRKG DPAVVVAASA AALEHHHHHH

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Macromolecule #2: Polyketide synthase

MacromoleculeName: Polyketide synthase / type: protein_or_peptide / ID: 2 / Details: S1816 is crosslinked to C553 in chain A / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Cercospora nicotianae (fungus)
Molecular weightTheoretical: 9.892837 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMDPSPNE IGTVWRDALK ILSEESGLTD EELTDDTSFA DVGVDSLMSL VITSRLRDEL DIDFPDRALF EECQTIFDLR KRFSGSTE

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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

Concentration0.27 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris(hydroxymethyl)aminomethan (TRIS)
50.0 mMNaClsodium chloride
2.5 mMC9H15O6Ptris(2-carboxyethyl)phosphine (TCEP)
GridModel: Quantifoil Lacey carbon grid / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK III
DetailsThe proteins have been crosslinked via site specific crosslinking.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 41.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 4.5 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 27707 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.8 µm / Nominal magnification: 105000
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

CTF correctionSoftware: (Name: CTFFIND (ver. 4.1), Gctf (ver. 1.06))
Startup modelType of model: INSILICO MODEL
In silico model: generated from 2D class averages using e2initialmodel.py (EMAN2)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2) / Number images used: 25107
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: OTHER / Details: Fourier space
Final 3D classificationNumber classes: 3 / Avg.num./class: 15000 / Software - Name: RELION (ver. 2)
FSC plot (resolution estimation)

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