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- EMDB-27758: Full-length E47K SPOP -

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Basic information

Entry
Database: EMDB / ID: EMD-27758
TitleFull-length E47K SPOP
Map dataFull map
Sample
  • Complex: SPOP E47K Mutant
    • Protein or peptide: Speckle-type POZ protein
Function / homology
Function and homology information


regulation of proteolysis / Cul3-RING ubiquitin ligase complex / molecular function inhibitor activity / Hedgehog 'on' state / protein polyubiquitination / proteasome-mediated ubiquitin-dependent protein catabolic process / nuclear speck / ubiquitin protein ligase binding / nucleoplasm / identical protein binding ...regulation of proteolysis / Cul3-RING ubiquitin ligase complex / molecular function inhibitor activity / Hedgehog 'on' state / protein polyubiquitination / proteasome-mediated ubiquitin-dependent protein catabolic process / nuclear speck / ubiquitin protein ligase binding / nucleoplasm / identical protein binding / nucleus / cytoplasm
Similarity search - Function
SPOP, C-terminal BACK domain / MATH domain / MATH/TRAF domain / MATH/TRAF domain profile. / meprin and TRAF homology / TRAF-like / BTB/POZ domain / BTB domain profile. / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily
Similarity search - Domain/homology
Speckle-type POZ protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsCuneo MJ / Mittag T / O'Flynn B / Lo YH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM112846 United States
CitationJournal: Mol Cell / Year: 2023
Title: Higher-order SPOP assembly reveals a basis for cancer mutant dysregulation.
Authors: Matthew J Cuneo / Brian G O'Flynn / Yu-Hua Lo / Nafiseh Sabri / Tanja Mittag /
Abstract: The speckle-type POZ protein (SPOP) functions in the Cullin3-RING ubiquitin ligase (CRL3) as a receptor for the recognition of substrates involved in cell growth, survival, and signaling. SPOP ...The speckle-type POZ protein (SPOP) functions in the Cullin3-RING ubiquitin ligase (CRL3) as a receptor for the recognition of substrates involved in cell growth, survival, and signaling. SPOP mutations have been attributed to the development of many types of cancers, including prostate and endometrial cancers. Prostate cancer mutations localize in the substrate-binding site of the substrate recognition (MATH) domain and reduce or prevent binding. However, most endometrial cancer mutations are dispersed in seemingly inconspicuous solvent-exposed regions of SPOP, offering no clear basis for their cancer-causing and peculiar gain-of-function properties. Herein, we present the first structure of SPOP in its oligomeric form, uncovering several new interfaces important for SPOP self-assembly and normal function. Given that many previously unaccounted-for cancer mutations are localized in these newly identified interfaces, we uncover molecular mechanisms underlying dysregulation of SPOP function, with effects ranging from gross structural changes to enhanced self-association, and heightened stability and activity.
History
DepositionAug 2, 2022-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateMar 22, 2023-
Current statusMar 22, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27758.png

Downloads & links

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Map

FileDownload / File: emd_27758.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 300 pix.
= 389.1 Å		1.3 Å/pix.
x 300 pix.
= 389.1 Å		1.3 Å/pix.
x 300 pix.
= 389.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.297 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.18
Minimum - Maximum-0.96389973 - 2.0189767
Average (Standard dev.)-0.0012630342 (±0.049057744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 389.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27758_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map

Fileemd_27758_half_map_1.map
AnnotationHalf Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map

Fileemd_27758_half_map_2.map
AnnotationHalf Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SPOP E47K Mutant

EntireName: SPOP E47K Mutant
Components
  • Complex: SPOP E47K Mutant
    • Protein or peptide: Speckle-type POZ protein

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Supramolecule #1: SPOP E47K Mutant

SupramoleculeName: SPOP E47K Mutant / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Speckle-type POZ protein

MacromoleculeName: Speckle-type POZ protein / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 42.183422 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MSRVPSPPPP AEMSSGPVAE SWCYTQIKVV KFSYMWTINN FSFCREKMGE VIKSSTFSSG ANDKLKWCLR VNPKGLDEES KDYLSLYLL LVSCPKSEVR AKFKFSILNA KGEETKAMES QRAYRFVQGK DWGFKKFIRR DFLLDEANGL LPDDKLTLFC E VSVVQDSV ...String:
MSRVPSPPPP AEMSSGPVAE SWCYTQIKVV KFSYMWTINN FSFCREKMGE VIKSSTFSSG ANDKLKWCLR VNPKGLDEES KDYLSLYLL LVSCPKSEVR AKFKFSILNA KGEETKAMES QRAYRFVQGK DWGFKKFIRR DFLLDEANGL LPDDKLTLFC E VSVVQDSV NISGQNTMNM VKVPECRLAD ELGGLWENSR FTDCCLCVAG QEFQAHKAIL AARSPVFSAM FEHEMEESKK NR VEINDVE PEVFKEMMCF IYTGKAPNLD KMADDLLAAA DKYALERLKV MCEDALCSNL SVENAAEILI LADLHSADQL KTQ AVDFIN YHASDVLETS GWKSMVVSHP HLVAEAYRSL ASAQCPFLGP PRKRLKQS

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5 / Details: 20 mM HEPES pH 7.5, 400 mM NaCl, 5 mM DTT
GridModel: C-flat-2/2 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 65.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3hqi

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 350000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3hqi

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8dws:
Full-length E47K SPOP

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