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- EMDB-40304: The local refinement map of a single subunit of a 12-LOX hexamer -

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Basic information

Entry
Database: EMDB / ID: EMD-40304
TitleThe local refinement map of a single subunit of a 12-LOX hexamer
Map dataThe map from a local refinement of a single subunit of a 12-LOX hexamer filtered according to local resolution
Sample
  • Complex: Dimeric human 12-Lipoxygenase
    • Protein or peptide: Polyunsaturated fatty acid lipoxygenase ALOX12
KeywordsLipoxygenase / platelets / lipid-modifying enzyme / lipid oxidation / OXIDOREDUCTASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.24 Å
AuthorsBlack KA / Mobbs JI / Venugopal H / Thal DM / Glukhova A
Funding support United States, 2 items
OrganizationGrant numberCountry
Other private
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM131835 United States
CitationJournal: Blood / Year: 2023
Title: Cryo-EM structures of human arachidonate 12S-lipoxygenase bound to endogenous and exogenous inhibitors.
Authors: Jesse I Mobbs / Katrina A Black / Michelle Tran / Wessel A C Burger / Hariprasad Venugopal / Theodore R Holman / Michael Holinstat / David M Thal / Alisa Glukhova /
Abstract: Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the ...Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms by which it affects platelet activation are not fully understood, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryo-electron microscopy to determine high-resolution structures (1.7-2.8 Å) of human 12-LOX. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may affect its catalytic activity and membrane association. We also identified different conformations within the 12-LOX dimer, which likely represent different time points in its catalytic cycle. Furthermore, we identified small molecules bound to 12-LOX. The active site of the 12-LOX tetramer was occupied by an endogenous 12-LOX inhibitor, a long-chain acyl coenzyme A. In addition, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase 1 clinical trial for the treatment of heparin-induced thrombocytopenia and received a fast-track designation by the Food and Drug Administration. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, their catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme.
History
DepositionApr 3, 2023-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40304.png

Downloads & links

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Map

FileDownload / File: emd_40304.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe map from a local refinement of a single subunit of a 12-LOX hexamer filtered according to local resolution
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.8
Minimum - Maximum-1.5408272 - 3.7939658
Average (Standard dev.)0.0013906568 (±0.052096877)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 262.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40304_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 from a local refinement of...

Fileemd_40304_half_map_1.map
AnnotationHalf map 1 from a local refinement of a single subunit of a 12-LOX hexamer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 from a local refinement of...

Fileemd_40304_half_map_2.map
AnnotationHalf map 2 from a local refinement of a single subunit of a 12-LOX hexamer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimeric human 12-Lipoxygenase

EntireName: Dimeric human 12-Lipoxygenase
Components
  • Complex: Dimeric human 12-Lipoxygenase
    • Protein or peptide: Polyunsaturated fatty acid lipoxygenase ALOX12

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Supramolecule #1: Dimeric human 12-Lipoxygenase

SupramoleculeName: Dimeric human 12-Lipoxygenase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Polyunsaturated fatty acid lipoxygenase ALOX12

MacromoleculeName: Polyunsaturated fatty acid lipoxygenase ALOX12 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MHHHHHHGRY RIRVATGAWL FSGSYNRVQL WLVGTRGEAE LELQLRPARG EEEEFDHDVA EDLGLLQFVR LRKHHWLVDD AWFCDRITV QGPGACAEVA FPCYRWVQGE DILSLPEGTA RLPGDNALDM FQKHREKELK DRQQIYCWAT WKEGLPLTIA A DRKDDLPP ...String:
MHHHHHHGRY RIRVATGAWL FSGSYNRVQL WLVGTRGEAE LELQLRPARG EEEEFDHDVA EDLGLLQFVR LRKHHWLVDD AWFCDRITV QGPGACAEVA FPCYRWVQGE DILSLPEGTA RLPGDNALDM FQKHREKELK DRQQIYCWAT WKEGLPLTIA A DRKDDLPP NMRFHEEKRL DFEWTLKAGA LEMALKRVYT LLSSWNCLED FDQIFWGQKS ALAEKVRQCW QDDELFSYQF LN GANPMLL RRSTSLPSRL VLPSGMEELQ AQLEKELQNG SLFEADFILL DGIPANVIRG EKQYLAAPLV MLKMEPNGKL QPM VIQIQP PSPSSPTPTL FLPSDPPLAW LLAKSWVRNS DFQLHEIQYH LLNTHLVAEV IAVATMRCLP GLHPIFKFLI PHIR YTMEI NTRARTQLIS DGGIFDKAVS TGGGGHVQLL RRAAAQLTYC SLCPPDDLAD RGLLGLPGAL YAHDALRLWE IIARY VEGI VHLFYQRDDI VKGDPELQAW CREITEVGLC QAQDRGFPVS FQSQSQLCHF LTMCVFTCTA QHAAINQGQL DWYAWV PNA PCTMRMPPPT TKEDVTMATV MGSLPDVRQA CLQMAISWHL SRRQPDMVPL GHHKEKYFSG PKPKAVLNQF RTDLEKL EK EITARNEQLD WPYEYLKPSC IENSVTI

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 215034
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT

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