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- EMDB-5651: Cryo-EM structure pentaketide-ACP4-PikAIII/C209A/delta ACP5 -

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Basic information

Entry
Database: EMDB / ID: 5651
TitleCryo-EM structure pentaketide-ACP4-PikAIII/C209A/delta ACP5
Map dataReconstruction of the 5th module from the pikromycin biosynthetic pathway (PikAIII) lacking the ACP domain with an N-terminal fusion to the ACP domain from module 4 of the pikromycin pathway (ACP4). The ACP4 domain was loaded with the pentaketide intermediate.
Samplepentaketide-ACP4-PikAIII/C209A/delta ACP5:
PikAIII / ligand
KeywordsType I polyketide synthase module
Function / homologyPolyketide synthase, acyl transferase domain / Malonyl-CoA ACP transacylase, ACP-binding / Acyl transferase domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase, N-terminal domain / ACP-like superfamily / Polyketide synthase, docking domain superfmaily / NAD(P)-binding domain superfamily / Ketoacyl-synthetase, C-terminal extension / Polyketide synthase, beta-ketoacyl synthase domain ...Polyketide synthase, acyl transferase domain / Malonyl-CoA ACP transacylase, ACP-binding / Acyl transferase domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase, N-terminal domain / ACP-like superfamily / Polyketide synthase, docking domain superfmaily / NAD(P)-binding domain superfamily / Ketoacyl-synthetase, C-terminal extension / Polyketide synthase, beta-ketoacyl synthase domain / Polyketide synthase, phosphopantetheine-binding domain / Erythronolide synthase docking / Beta-ketoacyl synthase, active site / Thiolase-like / Acyl transferase/acyl hydrolase/lysophospholipase / KR domain / Polyketide synthase, docking domain / Acyl transferase / Beta-ketoacyl synthase, C-terminal / Beta-ketoacyl synthase, N-terminal / Polyketide synthase, ketoreductase domain / Phosphopantetheine binding ACP domain / Phosphopantetheine attachment site / Acyl transferase domain superfamily / Ketoacyl-synthetase C-terminal extension / Phosphopantetheine attachment site. / Beta-ketoacyl synthases active site. / Carrier protein (CP) domain profile. / Beta-ketoacyl synthase, C-terminal domain / 10-deoxymethynolide synthase / narbonolide synthase / macrolide biosynthetic process / transferase activity, transferring acyl groups other than amino-acyl groups / phosphopantetheine binding / identical protein binding / Narbonolide/10-deoxymethynolide synthase PikA3, module 5
Function and homology information
SourceStreptomyces venezuelae (bacteria) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / 8.6 Å resolution
AuthorsDutta S / Whicher JR / Hansen DA / Hale WA / Chemler JA / Narayan AR / Hakansson K / Sherman DH / Smith JL / Skiniotis G
CitationJournal: Nature / Year: 2014
Title: Structure of a modular polyketide synthase.
Authors: Somnath Dutta / Jonathan R Whicher / Douglas A Hansen / Wendi A Hale / Joseph A Chemler / Grady R Congdon / Alison R H Narayan / Kristina Håkansson / David H Sherman / Janet L Smith / Georgios Skiniotis
DateDeposition: May 1, 2013 / Header (metadata) release: Jul 3, 2013 / Map release: Jun 25, 2014 / Last update: Oct 22, 2014

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 7.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_5651.map.gz (map file in CCP4 format, 27649 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
192 pix
2.24 Å/pix.
= 430.08 Å
192 pix
2.24 Å/pix.
= 430.08 Å
192 pix
2.24 Å/pix.
= 430.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.24 Å
Density
Contour Level:7.4 (by author), 7.4 (movie #1):
Minimum - Maximum-25.48913193 - 42.89436722
Average (Standard dev.)0E-8 (0.99999994)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions192192192
Origin-16-16-16
Limit175175175
Spacing192192192
CellA=B=C: 430.08002 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.242.242.24
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z430.080430.080430.080
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-16-16-16
NC/NR/NS192192192
D min/max/mean-25.48942.894-0.000

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Supplemental data

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Sample components

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Entire pentaketide-ACP4-PikAIII/C209A/delta ACP5

EntireName: pentaketide-ACP4-PikAIII/C209A/delta ACP5
Details: Sample was not frozen prior to loading on the grid. The sample was monodisperse.
Number of components: 2 / Oligomeric State: Dimer
MassTheoretical: 348 kDa / Experimental: 348 kDa / Measured by: Gel filtration chromatography.

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Component #1: protein, PikAIII

ProteinName: PikAIII / Oligomeric Details: Dimer / Number of Copies: 2 / Recombinant expression: Yes
MassTheoretical: 348 kDa / Experimental: 348 kDa
SourceSpecies: Streptomyces venezuelae (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET28b
External referencesUniProt: Narbonolide/10-deoxymethynolide synthase PikA3, module 5

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Component #2: ligand, pentaketide

LigandName: pentaketide / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 1
SourceSpecies: unidentified (others)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.1 mg/ml / Buffer solution: 50 mM HEPES, 100mM NaCl / pH: 7.4
Support filmGlow-discharged Quantifoil R2/200 mesh grid
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 89 K / Humidity: 100 %
Method: Blot for 1.5-2.0 seconds before plunging (3 microliter sample).

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Mar 3, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 22 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal), 66964 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 135,000 times magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3500 nm
Specimen HolderHolder: Oxford holder / Model: OTHER / Temperature: K ( 89 - 89 K)
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 365

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 43946 / Details: The particles were selected manually.
3D reconstructionAlgorithm: Cross-common lines / Software: EMAN1, EMAN2 / CTF correction: Each micrograph / Resolution: 8.6 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 2HG4
Chain ID: A, B
Modeling #2Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 2JU1
Modeling #3Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 2FR0

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