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- EMDB-26574: KS-AT di-domain of mycobacterial Pks13 with endogenous KS ligand bound -

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Basic information

Entry
Database: EMDB / ID: EMD-26574
TitleKS-AT di-domain of mycobacterial Pks13 with endogenous KS ligand bound
Map data
Sample
  • Complex: Mycobacterial polyketide synthase 13
    • Complex: Ketosynthase domainKetoacyl synthase
      • Protein or peptide: Polyketide synthase PKS13
    • Complex: Acyl transferase domain
  • Ligand: UNKNOWN LIGAND
  • Ligand: water
Keywordsmycolic acid synthesis / ketosynthase / acyltransferase / multi-domain assembly / BIOSYNTHETIC PROTEIN
Function / homology
Function and homology information


6-deoxyerythronolide-B synthase / erythronolide synthase activity / lipid biosynthetic process / phosphopantetheine binding
Similarity search - Function
Thioesterase / Thioesterase 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 ...Thioesterase / Thioesterase 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 / Ketosynthase family 3 (KS3) domain profile. / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase / 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
Polyketide synthase PKS13
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.94 Å
AuthorsKim SK / Dickinson MS / Finer-Moore JS / Rosenberg OS / Stroud RM
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01 AI095208 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI128214 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM24485 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13.
Authors: Sun Kyung Kim / Miles Sasha Dickinson / Janet Finer-Moore / Ziqiang Guan / Robyn M Kaake / Ignacia Echeverria / Jen Chen / Ernst H Pulido / Andrej Sali / Nevan J Krogan / Oren S Rosenberg / Robert M Stroud /
Abstract: The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug ...The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified from mycobacteria under normal growth conditions, captured with native substrates. Structures define the ketosynthase (KS), linker and acyl transferase (AT) domains at 1.8 Å resolution and two alternative locations of the N-terminal acyl carrier protein. These structures suggest intermediate states on the pathway for substrate delivery to the KS domain. Other domains, visible at lower resolution, are flexible relative to the KS-AT core. The chemical structures of three bound endogenous long-chain fatty acid substrates were determined by electrospray ionization mass spectrometry.
History
DepositionMar 31, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateJun 14, 2023-
Current statusJun 14, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26574.png

Downloads & links

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Map

FileDownload / File: emd_26574.map.gz / Format: CCP4 / Size: 219.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.333
Minimum - Maximum-0.26222926 - 1.5503584
Average (Standard dev.)-0.00013110462 (±0.039638605)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions386386386
Spacing386386386
CellA=B=C: 322.31 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: density-modified, sharpened map

Fileemd_26574_additional_1.map
Annotationdensity-modified, sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DeepEMhancer sharpened map contour at 0.268

Fileemd_26574_additional_2.map
AnnotationDeepEMhancer sharpened map contour at 0.268
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26574_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26574_half_map_2.map
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Sample components

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Entire : Mycobacterial polyketide synthase 13

EntireName: Mycobacterial polyketide synthase 13
Components
  • Complex: Mycobacterial polyketide synthase 13
    • Complex: Ketosynthase domainKetoacyl synthase
      • Protein or peptide: Polyketide synthase PKS13
    • Complex: Acyl transferase domain
  • Ligand: UNKNOWN LIGAND
  • Ligand: water

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Supramolecule #1: Mycobacterial polyketide synthase 13

SupramoleculeName: Mycobacterial polyketide synthase 13 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: The gene for Mycobacterium smegmatis polyketide synthase 13 (Pks13) was tagged with TEV-cleavable GFP at its C-terminus and purified from its natural source with anti-GFP nanobody beads. GFP ...Details: The gene for Mycobacterium smegmatis polyketide synthase 13 (Pks13) was tagged with TEV-cleavable GFP at its C-terminus and purified from its natural source with anti-GFP nanobody beads. GFP was cleaved to yield the full-length Pks13.
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Supramolecule #2: Ketosynthase domain

SupramoleculeName: Ketosynthase domain / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Linked to acyl transferase domain at the C-terminus
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Supramolecule #3: Acyl transferase domain

SupramoleculeName: Acyl transferase domain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1 / Details: linked to ketosynthase domain at the N-terminus
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Macromolecule #1: Polyketide synthase PKS13

MacromoleculeName: Polyketide synthase PKS13 / type: protein_or_peptide / ID: 1
Details: The gene for M. smegmatis polyketide synthase 13 (Pks13) was tagged with TEV-cleavable GFP at its C-terminus and purified from its natural source with anti-GFP nanobody beads. GFP was ...Details: The gene for M. smegmatis polyketide synthase 13 (Pks13) was tagged with TEV-cleavable GFP at its C-terminus and purified from its natural source with anti-GFP nanobody beads. GFP was cleaved to yield the full-length Pks13.
Number of copies: 2 / Enantiomer: LEVO / EC number: 6-deoxyerythronolide-B synthase
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Strain: ATCC 700084 / mc(2)155
Molecular weightTheoretical: 198.633359 KDa
SequenceString: MDENHSDSNL PEGTEPAGRP APRTDMTVNE MREWLRNWVA NATGQSADAI DESTPMVELG LSSRDAVAMA SDIEDLTGVT LTATVAFRH PTIESLATVI IEGEPEPEPY DEDEDWSRTR DVEDIAIVGV ATRFPGDLNT PDEMWEALLE GKDCVTDLPE D RWTEFLDE ...String:
MDENHSDSNL PEGTEPAGRP APRTDMTVNE MREWLRNWVA NATGQSADAI DESTPMVELG LSSRDAVAMA SDIEDLTGVT LTATVAFRH PTIESLATVI IEGEPEPEPY DEDEDWSRTR DVEDIAIVGV ATRFPGDLNT PDEMWEALLE GKDCVTDLPE D RWTEFLDE PRIAERVKKA RTRGGYLTDI KGFDSEFFAL SKMEADNIDP QQRMALELTW EALEHARIPA SSLRGESVGV YI GSSTNDY SFLAMSDPSI AHPYAITGTA SSIIANRVSY FYDFRGPSVA VDTACSSSLV ATHQGVQALR AGEADVAIVG GVN ALVTPL VTVGFDEVGG VLAPDGRIKS FSSDADGYAR SEGGGMLVLK RISDARRDGD QILAVIAGSA VNHDGRSNGL LAPN PDAQA EVLRKAYKDA GINPRDVDYI EAHGTGTILG DPIEADALGR IVGKGRPADK PALLGAVKSN LGHLESAAGA ASLAK MTLA LANDKLPPSI NYAGPNPYID FEKERLKVND TVSDWPRYSG KAIAGVSGFG FGGANAHVVM REVLAGDLVE PEPEPE PEA KPEKSEADAV YVGGVRMDEY GEFIDEDEPA EGGDAYPSYD EDSYELPGIT EAAQRLLEQA REELEAKEAE EPTKQLV PL AVSAFLTSRK RQAAAELADW IDSPEGRASS LESIGRSLSR RNHGRSRAVV LAHDHDEAIK GLRALAEGKQ HPSVLSAD G PVTNGPVWVL AGFGAQHRKM GKSLYLRNEV FAEWINKVDA LIQDERGYSI LELILDDNVD YTDATCEYPI EVVQLVIFA IQIALGELLR HHGAKPAAVV GQSLGEAAAS YFAGGLSLAD ATRTICSRSH LMGEGEAMLF GEYIRLMALV EYSADEIKTV FSDYPDLEV CVYAAPTQTV IGGPPDQVDA IIARAESEGK FARKFQTKGA SHTQQMDPLL GELAAELQGI EPKPLTTGYF S TVHEGTFI RPGSAPIHDV DYWKKGLRHS VYFTQGIRNA VDNGHTTFLE LAPNPVALMQ VGLTTASAGL HDAQLIATLA RK QDEVESM ISAMAQLYVH GHDLDFRTLF PRRSKGLAGA LDFANIPPTR FKRKEHWLPA HFTGDSSAVM PGNHVATPDG RHV WEFVPR GKTDLAALVK AAAAQVLPDA KLAAFEQRAV PADNARLVTT LTRHPGGATV QVHARVEESF TLVYDAIVAR ANGA GVTAL PVAVGAGVAV SGDVAGEGAG ASVIEDDEPD AEILQDNLTA GAGMGADFQK WDPNSGETIG QRLGTIVGAA MGYEP EDLP WEVPLIELGL DSLMAVRIKN RVEYDFDLPP IQLTAVRDAN LYNVEELIRY AIEHRDEVEQ IAESQKGKTA EEIAAE QSE LLGGASTVAE LEAKLAEAGH PLAAKDSEDS ENSEDNAAGA AAAAEASAVE GLEIPPPPTD PTGPGGAPIP PPPSDPS GP AQAASATDAP AGTVNKATAA AAAAKVLTQE AVTEALGADV PPRDAAERVT FATWAIVTGK SPGGIFNELP TVSEETAK K MAERLSERAE GTITVEDVLG AKTIEGLATI VREQLEEGVV DGFVRTLRPP KEGSNAVPLF VFHPAGGSTV VYEPLMKRL PADVPVYGLE RVEGSIEERA AEYVPKLLEM HKGPFVLAGW SLGGALAYAC AIGLKQSGAD VRFVGLIDTV LPGEPIDQSK EGMRARWDR YARFAERTFN VEIPAIPYEE LEKLDDEGQV KYVLEIVKES GVQIPGGIIE HQRTSYLDNR ALDTVDIKPY D GHVTLYMA DRYHDDAIVF EPAYATRKPD GGWGSFVSDL EVVHIGGEHI QAIDEPYIAK VGAHMSEALN RIEAQASKED GA KSKSTSE NLYFQ

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Macromolecule #2: UNKNOWN LIGAND

MacromoleculeName: UNKNOWN LIGAND / type: ligand / ID: 2
Details: The fatty acid ligand designated as UNL (unknown ligand) is a divided population of fatty acids of formula C55H106O2 and C40H78O2 as confirmed by mass spectrometry.
Number of copies: 2 / Formula: UNL
Molecular weightTheoretical: 312.53 Da
Chemical component information


ChemComp, No image

ChemComp-UNL:
Unknown ligand

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 390 / 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:
ConcentrationName
50.0 mMTris
150.0 mMNaClSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV
Details: Full length pks13 was concentrated to 1.5 mg mL-1 for cryo-EM grid preparation. 4.5 uL of sample was applied to freshly glow discharged holey carbon on gold R1.2/1.3 300 mesh Quantifoil ...Details: Full length pks13 was concentrated to 1.5 mg mL-1 for cryo-EM grid preparation. 4.5 uL of sample was applied to freshly glow discharged holey carbon on gold R1.2/1.3 300 mesh Quantifoil grids and blotted for 9 s with Whatman 1 filter paper at max humidity and 10oC in a FEI Mark IV Vitrobot, before vitrification in liquid nitrogen-cooled liquid ethane..

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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 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 7567 / Average exposure time: 5.9 sec. / Average electron dose: 45.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4400000
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.12)
Final 3D classificationNumber classes: 5 / Avg.num./class: 200000 / Software - Name: cryoSPARC (ver. 2.12)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.12)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.94 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.12) / Number images used: 1200000
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7uk4:
KS-AT di-domain of mycobacterial Pks13 with endogenous KS ligand bound

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