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- EMDB-27003: KS-AT domains of mycobacterial Pks13 with inward AT conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-27003
TitleKS-AT domains of mycobacterial Pks13 with inward AT conformation
Map data
Sample
  • Complex: Mycobacterial polyketide synthase 13 KS-AT domains with inward AT conformation
    • Protein or peptide: Polyketide synthase PKS13
  • Ligand: UNKNOWN LIGAND
Keywordsmycolic acid synthesis / ketosynthase / acyltransferase / multi-domain assembly / BIOSYNTHETIC PROTEIN
Function / homology
Function and homology information


6-deoxyerythronolide-B synthase / erythronolide synthase activity / DIM/DIP cell wall layer assembly / fatty acid synthase activity / phosphopantetheine binding / fatty acid biosynthetic process / plasma membrane / cytoplasm
Similarity search - Function
: / Thioesterase / Thioesterase domain / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Malonyl-CoA ACP transacylase, ACP-binding / : / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. ...: / Thioesterase / Thioesterase domain / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / 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 / Ketosynthase family 3 (KS3) domain profile. / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / Polyketide synthase, beta-ketoacyl synthase domain / 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: 3.0 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
DepositionMay 17, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27003.png

Downloads & links

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Map

FileDownload / File: emd_27003.map.gz / Format: CCP4 / Size: 219.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
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AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 386 pix.
= 322.31 Å		0.84 Å/pix.
x 386 pix.
= 322.31 Å		0.84 Å/pix.
x 386 pix.
= 322.31 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0126
Minimum - Maximum-0.013924925 - 0.05428307
Average (Standard dev.)-0.000063613705 (±0.0015358019)
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: DeepEMhancer modified map recommended contour at 0.0292

Fileemd_27003_additional_1.map
AnnotationDeepEMhancer modified map recommended contour at 0.0292
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Supplemental map: emd 27003 additional 2.map

Fileemd_27003_additional_2.map
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Half map: #2

Fileemd_27003_half_map_1.map
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Half map: #1

Fileemd_27003_half_map_2.map
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Sample components

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Entire : Mycobacterial polyketide synthase 13 KS-AT domains with inward AT...

EntireName: Mycobacterial polyketide synthase 13 KS-AT domains with inward AT conformation
Components
  • Complex: Mycobacterial polyketide synthase 13 KS-AT domains with inward AT conformation
    • Protein or peptide: Polyketide synthase PKS13
  • Ligand: UNKNOWN LIGAND

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Supramolecule #1: Mycobacterial polyketide synthase 13 KS-AT domains with inward AT...

SupramoleculeName: Mycobacterial polyketide synthase 13 KS-AT domains with inward AT conformation
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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Macromolecule #1: Polyketide synthase PKS13

MacromoleculeName: Polyketide synthase PKS13 / type: protein_or_peptide / ID: 1 / 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: 194.668203 KDa
SequenceString: MTVNEMREWL RNWVANATGQ SADAIDESTP MVELGLSSRD AVAMASDIED LTGVTLTATV AFRHPTIESL ATVIIEGEPE PEPYDEDED WSRTRDVEDI AIVGVATRFP GDLNTPDEMW EALLEGKDCV TDLPEDRWTE FLDEPRIAER VKKARTRGGY L TDIKGFDS ...String:
MTVNEMREWL RNWVANATGQ SADAIDESTP MVELGLSSRD AVAMASDIED LTGVTLTATV AFRHPTIESL ATVIIEGEPE PEPYDEDED WSRTRDVEDI AIVGVATRFP GDLNTPDEMW EALLEGKDCV TDLPEDRWTE FLDEPRIAER VKKARTRGGY L TDIKGFDS EFFALSKMEA DNIDPQQRMA LELTWEALEH ARIPASSLRG ESVGVYIGSS TNDYSFLAMS DPSIAHPYAI TG TASSIIA NRVSYFYDFR GPSVAVDTAC SSSLVATHQG VQALRAGEAD VAIVGGVNAL VTPLVTVGFD EVGGVLAPDG RIK SFSSDA DGYARSEGGG MLVLKRISDA RRDGDQILAV IAGSAVNHDG RSNGLLAPNP DAQAEVLRKA YKDAGINPRD VDYI EAHGT GTILGDPIEA DALGRIVGKG RPADKPALLG AVKSNLGHLE SAAGAASLAK MTLALANDKL PPSINYAGPN PYIDF EKER LKVNDTVSDW PRYSGKAIAG VSGFGFGGAN AHVVMREVLA GDLVEPEPEP EPEAKPEKSE ADAVYVGGVR MDEYGE FID EDEPAEGGDA YPSYDEDSYE LPGITEAAQR LLEQAREELE AKEAEEPTKQ LVPLAVSAFL TSRKRQAAAE LADWIDS PE GRASSLESIG RSLSRRNHGR SRAVVLAHDH DEAIKGLRAL AEGKQHPSVL SADGPVTNGP VWVLAGFGAQ HRKMGKSL Y LRNEVFAEWI NKVDALIQDE RGYSILELIL DDNVDYTDAT CEYPIEVVQL VIFAIQIALG ELLRHHGAKP AAVVGQSLG EAAASYFAGG LSLADATRTI CSRSHLMGEG EAMLFGEYIR LMALVEYSAD EIKTVFSDYP DLEVCVYAAP TQTVIGGPPD QVDAIIARA ESEGKFARKF QTKGASHTQQ MDPLLGELAA ELQGIEPKPL TTGYFSTVHE GTFIRPGSAP IHDVDYWKKG L RHSVYFTQ GIRNAVDNGH TTFLELAPNP VALMQVGLTT ASAGLHDAQL IATLARKQDE VESMISAMAQ LYVHGHDLDF RT LFPRRSK GLAGALDFAN IPPTRFKRKE HWLPAHFTGD SSAVMPGNHV ATPDGRHVWE FVPRGKTDLA ALVKAAAAQV LPD AKLAAF EQRAVPADNA RLVTTLTRHP GGATVQVHAR VEESFTLVYD AIVARANGAG VTALPVAVGA GVAVSGDVAG EGAG ASVIE DDEPDAEILQ DNLTAGAGMG ADFQKWDPNS GETIGQRLGT IVGAAMGYEP EDLPWEVPLI ELGLDSLMAV RIKNR VEYD FDLPPIQLTA VRDANLYNVE ELIRYAIEHR DEVEQIAESQ KGKTAEEIAA EQSELLGGAS TVAELEAKLA EAGHPL AAK DSEDSENSED NAAGAAAAAE ASAVEGLEIP PPPTDPTGPG GAPIPPPPSD PSGPAQAASA TDAPAGTVNK ATAAAAA AK VLTQEAVTEA LGADVPPRDA AERVTFATWA IVTGKSPGGI FNELPTVSEE TAKKMAERLS ERAEGTITVE DVLGAKTI E GLATIVREQL EEGVVDGFVR TLRPPKEGSN AVPLFVFHPA GGSTVVYEPL MKRLPADVPV YGLERVEGSI EERAAEYVP KLLEMHKGPF VLAGWSLGGA LAYACAIGLK QSGADVRFVG LIDTVLPGEP IDQSKEGMRA RWDRYARFAE RTFNVEIPAI PYEELEKLD DEGQVKYVLE IVKESGVQIP GGIIEHQRTS YLDNRALDTV DIKPYDGHVT LYMADRYHDD AIVFEPAYAT R KPDGGWGS FVSDLEVVHI GGEHIQAIDE PYIAKVGAHM SEALNRIEAQ ASKEDGAK

UniProtKB: Polyketide synthase PKS13

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Macromolecule #2: UNKNOWN LIGAND

MacromoleculeName: UNKNOWN LIGAND / type: ligand / ID: 2 / 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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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 mMNaCl
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
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4400000
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 123116
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.12)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.12)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8cuz:
KS-AT domains of mycobacterial Pks13 with inward AT conformation

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