[English] 日本語
Yorodumi
- EMDB-5666: Cryo-EM structure of beta-hydroxyhexaketide-PikAIII conformation 2 -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-5666
TitleCryo-EM structure of beta-hydroxyhexaketide-PikAIII conformation 2
Map dataReconstruction of the 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide.
Sample
  • Sample: The 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide
  • Protein or peptide: PikAIII
  • Ligand: NADPHNicotinamide adenine dinucleotide phosphate
  • Ligand: Methylmalonyl-CoA
  • Ligand: Thiophenol-pentaketide
KeywordsType I polyketide synthase module
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 / identical protein binding
Similarity search - Function
Polyketide synthase, docking domain superfmaily / Polyketide synthase, docking domain / Erythronolide synthase docking domain / PKS_PP_betabranch / Polyketide synthase, ketoreductase domain / KR domain / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Malonyl-CoA ACP transacylase, ACP-binding / PKS_KR ...Polyketide synthase, docking domain superfmaily / Polyketide synthase, docking domain / Erythronolide synthase docking domain / PKS_PP_betabranch / Polyketide synthase, ketoreductase domain / KR domain / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Malonyl-CoA ACP transacylase, ACP-binding / PKS_KR / Acyl transferase domain superfamily / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase/acyl hydrolase/lysophospholipase / Ketosynthase family 3 (KS3) domain profile. / 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 / Phosphopantetheine attachment site. / Thiolase-like / 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 PikA3, module 5
Similarity search - Component
Biological speciesStreptomyces venezuelae (bacteria) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.0 Å
AuthorsWhicher JR / Dutta S / Hansen DA / Hale WA / Chemler JA / Narayan AR / Hakansson K / Sherman DH / Smith JL / Skiniotis G
CitationJournal: Nature / Year: 2014
Title: Structural rearrangements of a polyketide synthase module during its catalytic cycle.
Authors: Jonathan R Whicher / Somnath Dutta / Douglas A Hansen / Wendi A Hale / Joseph A Chemler / Annie M Dosey / Alison R H Narayan / Kristina Håkansson / David H Sherman / Janet L Smith / Georgios Skiniotis /
Abstract: The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical ...The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the β-keto intermediate, and after β-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.
History
DepositionMay 2, 2013-
Header (metadata) releaseJul 3, 2013-
Map releaseJun 25, 2014-
UpdateOct 22, 2014-
Current statusOct 22, 2014Processing site: RCSB / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 6.8
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5666.gif

Downloads & links

-
Map

FileDownload / File: emd_5666.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide.
Voxel sizeX=Y=Z: 2.24 Å
Density
Contour LevelBy AUTHOR: 6.8 / Movie #1: 6.8
Minimum - Maximum-10.71022892 - 31.39071083
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-16-16-16
Dimensions192192192
Spacing192192192
CellA=B=C: 430.08002 Å
α=β=γ: 90.0 °

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-10.71031.3910.000

-
Supplemental data

-
Sample components

-
Entire : The 5th module from the pikromycin biosynthetic pathway (PikAIII)...

EntireName: The 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide
Components
  • Sample: The 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide
  • Protein or peptide: PikAIII
  • Ligand: NADPHNicotinamide adenine dinucleotide phosphate
  • Ligand: Methylmalonyl-CoA
  • Ligand: Thiophenol-pentaketide

-
Supramolecule #1000: The 5th module from the pikromycin biosynthetic pathway (PikAIII)...

SupramoleculeName: The 5th module from the pikromycin biosynthetic pathway (PikAIII) incubated with NADPH, methylmalonyl-CoA, and thiophenol-pentaketide
type: sample / ID: 1000
Details: Sample was not frozen prior to loading on the grid. The sample was monodisperse.
Oligomeric state: Dimer / Number unique components: 4
Molecular weightExperimental: 328 KDa / Theoretical: 328 KDa / Method: Gel filtration chromatography

-
Macromolecule #1: PikAIII

MacromoleculeName: PikAIII / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Streptomyces venezuelae (bacteria)
Molecular weightExperimental: 328 KDa / Theoretical: 328 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET28b
SequenceUniProtKB: Narbonolide/10-deoxymethynolide synthase PikA3, module 5

-
Macromolecule #2: NADPH

MacromoleculeName: NADPH / type: ligand / ID: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

-
Macromolecule #3: Methylmalonyl-CoA

MacromoleculeName: Methylmalonyl-CoA / type: ligand / ID: 3 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

-
Macromolecule #4: Thiophenol-pentaketide

MacromoleculeName: Thiophenol-pentaketide / type: ligand / ID: 4 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.4 / Details: 50 mM HEPES, 100mM NaCl
GridDetails: Glow-discharged Quantifoil R2/200 mesh grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 89 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 1.5-2.0 seconds before plunging.

-
Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 66964 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: OTHER
TemperatureMin: 89 K / Max: 89 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 135,000 times magnification.
DateOct 12, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 387 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

-
Image processing

CTF correctionDetails: Each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1, EMAN2 / Number images used: 12939
DetailsThe particles were selected manually.

-
Atomic model buiding 1

Initial modelPDB ID:

2hg4


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

-
Atomic model buiding 2

Initial modelPDB ID:

2fr0

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

-
Atomic model buiding 3

Initial modelPDB ID:

2ju1

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more