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- EMDB-21811: EM structure of CtBP2 with a minimal dehydrogenase domain of CtBP2 -

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Basic information

Entry
Database: EMDB / ID: EMD-21811
TitleEM structure of CtBP2 with a minimal dehydrogenase domain of CtBP2
Map dataCryoEM map of CtBP2 with the minimal dehydrogenase domain, CtBP31-364
Sample
  • Complex: Complex of CtBP2 with four NADH molecules
    • Protein or peptide: C-terminal-binding protein 2
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
KeywordsTranscriptional corepression / Cancer / Gene repression / metabolic sensor / GENE REGULATION
Function / homology
Function and homology information


positive regulation of retinoic acid receptor signaling pathway / structural constituent of presynaptic active zone / Signaling by TCF7L2 mutants / Repression of WNT target genes / synaptic vesicle docking / photoreceptor ribbon synapse / presynaptic active zone cytoplasmic component / nuclear retinoic acid receptor binding / presynaptic cytosol / Sensory processing of sound by inner hair cells of the cochlea ...positive regulation of retinoic acid receptor signaling pathway / structural constituent of presynaptic active zone / Signaling by TCF7L2 mutants / Repression of WNT target genes / synaptic vesicle docking / photoreceptor ribbon synapse / presynaptic active zone cytoplasmic component / nuclear retinoic acid receptor binding / presynaptic cytosol / Sensory processing of sound by inner hair cells of the cochlea / oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor / white fat cell differentiation / GABA-ergic synapse / transcription repressor complex / cellular response to leukemia inhibitory factor / viral genome replication / transcription corepressor binding / transcription coregulator binding / transcription corepressor activity / NAD binding / DNA-binding transcription factor binding / transcription coactivator activity / negative regulation of cell population proliferation / negative regulation of DNA-templated transcription / glutamatergic synapse / chromatin binding / protein-containing complex binding / regulation of transcription by RNA polymerase II / protein kinase binding / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / identical protein binding / nucleus
Similarity search - Function
C-terminal binding protein / D-isomer specific 2-hydroxyacid dehydrogenases signature 3. / D-isomer specific 2-hydroxyacid dehydrogenase, NAD-binding domain conserved site / D-isomer specific 2-hydroxyacid dehydrogenase, catalytic domain / D-isomer specific 2-hydroxyacid dehydrogenase, catalytic domain / D-isomer specific 2-hydroxyacid dehydrogenase, NAD-binding domain / D-isomer specific 2-hydroxyacid dehydrogenase, NAD binding domain / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
C-terminal-binding protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsJecrois AM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM119014 United States
CitationJournal: Structure / Year: 2021
Title: Cryo-EM structure of CtBP2 confirms tetrameric architecture.
Authors: Anne M Jecrois / M Michael Dcona / Xiaoyan Deng / Dipankar Bandyopadhyay / Steven R Grossman / Celia A Schiffer / William E Royer /
Abstract: C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis, and metastasis. NADH-dependent ...C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis, and metastasis. NADH-dependent CtBP activation has been implicated in multiple types of cancer and poor patient prognosis. Central to understanding activation of CtBP in oncogenesis is uncovering how NADH triggers protein assembly, what level of assembly occurs, and if oncogenic activity depends upon such assembly. Here, we present the cryoelectron microscopic structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is tetrameric. The physiological relevance of the observed tetramer was demonstrated in cell culture, showing that CtBP tetramer-destabilizing mutants are defective for cell migration, transcriptional repression of E-cadherin, and activation of TIAM1. Together with our cryoelectron microscopy studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.
History
DepositionApr 17, 2020-
Header (metadata) releaseDec 2, 2020-
Map releaseDec 2, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.042
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.042
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wkw
  • Surface level: 0.042
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21811.png

Downloads & links

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Map

FileDownload / File: emd_21811.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM map of CtBP2 with the minimal dehydrogenase domain, CtBP31-364
Voxel sizeX=Y=Z: 0.87 Å
Density
Contour LevelBy AUTHOR: 0.042 / Movie #1: 0.042
Minimum - Maximum-0.09483134 - 0.20145601
Average (Standard dev.)0.0007019255 (±0.0073046074)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 222.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.870.870.87
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z222.720222.720222.720
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0950.2010.001

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Supplemental data

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Half map: EM structure of CtBP2 Half map

Fileemd_21811_half_map_1.map
AnnotationEM structure of CtBP2 Half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM structure of CtBP2 Half map

Fileemd_21811_half_map_2.map
AnnotationEM structure of CtBP2 Half 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 : Complex of CtBP2 with four NADH molecules

EntireName: Complex of CtBP2 with four NADH molecules
Components
  • Complex: Complex of CtBP2 with four NADH molecules
    • Protein or peptide: C-terminal-binding protein 2
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: Complex of CtBP2 with four NADH molecules

SupramoleculeName: Complex of CtBP2 with four NADH molecules / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 190 KDa

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Macromolecule #1: C-terminal-binding protein 2

MacromoleculeName: C-terminal-binding protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 36.620715 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: RPLVALLDGR DCTVEMPILK DLATVAFCDA QSTQEIHEKV LNEAVGAMMY HTITLTREDL EKFKALRVIV RIGSGYDNVD IKAAGELGI AVCNIPSAAV EETADSTICH ILNLYRRNTW LYQALREGTR VQSVEQIREV ASGAARIRGE TLGLIGFGRT G QAVAVRAK ...String:
RPLVALLDGR DCTVEMPILK DLATVAFCDA QSTQEIHEKV LNEAVGAMMY HTITLTREDL EKFKALRVIV RIGSGYDNVD IKAAGELGI AVCNIPSAAV EETADSTICH ILNLYRRNTW LYQALREGTR VQSVEQIREV ASGAARIRGE TLGLIGFGRT G QAVAVRAK AFGFSVIFYD PYLQDGIERS LGVQRVYTLQ DLLYQSDCVS LHCNLNEHNH HLINDFTIKQ MRQGAFLVNA AR GGLVDEK ALAQALKEGR IRGAALDVHE SEPFSFAQGP LKDAPNLICT PHTAWYSEQA SLEMREAAAT EIRRAITGRI PES LRNCVN KEFF

UniProtKB: C-terminal-binding protein 2

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Macromolecule #2: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 4 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM / Nicotinamide adenine dinucleotide

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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

Concentration0.25 mg/mL
BufferpH: 7.5
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
Details: Grid was washed in Ethyl acetate prior to glow-discharge.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 278.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time: 4s Blotting force: 8 Wait time: 0.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3405 / Average exposure time: 1.7 sec. / Average electron dose: 37.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 485473 / Details: Particles were automatically picked in cisTEM.
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM
Final 3D classificationNumber classes: 3 / Software - Name: RSRef (ver. 3.0.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.2) / Number images used: 112919
DetailsImages were beam-motion corrected and aligned.

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Atomic model buiding 1

Initial modelPDB ID:

4u6q


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6wkw:
EM structure of CtBP2 with a minimal dehydrogenase domain of CtBP2

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