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- EMDB-21382: Negative stain EM map of an MTA-HDAC-MBD complex -

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Basic information

Entry
Database: EMDB / ID: EMD-21382
TitleNegative stain EM map of an MTA-HDAC-MBD complex
Map dataNegative stain EM map of an MTA-HDAC-MBD complex.
Sample
  • Complex: MHm complex
    • Complex: Metastasis-associated protein MTA2,
      • Protein or peptide: Metastasis-associated protein MTA2
      • Protein or peptide: Methyl-CpG-binding protein MBD3/GATA zinc finger domain-containing protein 2A fusion
    • Complex: Histone deacetylase 1
      • Protein or peptide: Histone deacetylase 1
Biological speciesMus musculus (house mouse) / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 29.0 Å
AuthorsSilva APG / Low JKK / Tabar MS / Torrado M / Schmidberger JW / Brillault L / Landsberg MJ / Mackay JP
Funding support Australia, 1 items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)APP1012161 Australia
CitationJournal: Cell Rep / Year: 2020
Title: The Nucleosome Remodeling and Deacetylase Complex Has an Asymmetric, Dynamic, and Modular Architecture.
Authors: Jason K K Low / Ana P G Silva / Mehdi Sharifi Tabar / Mario Torrado / Sarah R Webb / Benjamin L Parker / Maryam Sana / Callum Smits / Jason W Schmidberger / Lou Brillault / Matthew J Jackman ...Authors: Jason K K Low / Ana P G Silva / Mehdi Sharifi Tabar / Mario Torrado / Sarah R Webb / Benjamin L Parker / Maryam Sana / Callum Smits / Jason W Schmidberger / Lou Brillault / Matthew J Jackman / David C Williams / Gerd A Blobel / Sandra B Hake / Nicholas E Shepherd / Michael J Landsberg / Joel P Mackay /
Abstract: The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native ...The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics. The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.
History
DepositionFeb 13, 2020-
Header (metadata) releaseFeb 26, 2020-
Map releaseDec 16, 2020-
UpdateDec 16, 2020-
Current statusDec 16, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21382.png

Downloads & links

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Map

FileDownload / File: emd_21382.map.gz / Format: CCP4 / Size: 12.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM map of an MTA-HDAC-MBD complex.
Voxel sizeX=Y=Z: 2.79 Å
Density
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.06063687 - 0.45139435
Average (Standard dev.)0.0022417589 (±0.021404171)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions148148148
Spacing148148148
CellA=B=C: 412.91998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.792.792.79
M x/y/z148148148
origin x/y/z0.0000.0000.000
length x/y/z412.920412.920412.920
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS148148148
D min/max/mean-0.0610.4510.002

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Supplemental data

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Sample components

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Entire : MHm complex

EntireName: MHm complex
Components
  • Complex: MHm complex
    • Complex: Metastasis-associated protein MTA2,
      • Protein or peptide: Metastasis-associated protein MTA2
      • Protein or peptide: Methyl-CpG-binding protein MBD3/GATA zinc finger domain-containing protein 2A fusion
    • Complex: Histone deacetylase 1
      • Protein or peptide: Histone deacetylase 1

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Supramolecule #1: MHm complex

SupramoleculeName: MHm complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Complex formed by HA-MTA2(1-429), HA-HDAC1 and FLAG-MBD3cc that were recombinantly expressed in HEK293 cells.
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293
Molecular weightExperimental: 300 KDa

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Supramolecule #2: Metastasis-associated protein MTA2,

SupramoleculeName: Metastasis-associated protein MTA2, / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293

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Supramolecule #3: Histone deacetylase 1

SupramoleculeName: Histone deacetylase 1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: Metastasis-associated protein MTA2

MacromoleculeName: Metastasis-associated protein MTA2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAANMYRVGD YVYFENSSSN PYLVRRIEEL NKTANGNVEA KVVCLFRRRD ISSSLNSLAD SNAREFEEE SKQPGVSEQQ RHQLKHRELF LSRQFESLPA THIRGKCSVT LLNETDILNQ Y LDKEDCFF YSLVFDPVQK TLLADQGEIR VGCKFQAEIP DRLAEGESDN ...String:
MAANMYRVGD YVYFENSSSN PYLVRRIEEL NKTANGNVEA KVVCLFRRRD ISSSLNSLAD SNAREFEEE SKQPGVSEQQ RHQLKHRELF LSRQFESLPA THIRGKCSVT LLNETDILNQ Y LDKEDCFF YSLVFDPVQK TLLADQGEIR VGCKFQAEIP DRLAEGESDN RNQQKMEMKV WD PDNPLTD RQIDQFLVVA RAVGTFARAL DCSSSIRQPS LHMSAAAASR DITLFHAMDT LQR NGYDLA KAMSTLVPQG GPVLCRDEME EWSASEAMLF EEALEKYGKD FNDIRQDFLP WKSL ASIVQ FYYMWKTTDR YIQQKRLKAA EADSKLKQVY IPTYTKPNPN QIISVGSKPG MNGAG FQKG LTCESCHTTQ SAQWYAWGPP NMQCRLCASC WIYWKKYGGL KTPTQLEGAA RGTTEP HSR GHLSRP

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Macromolecule #2: Histone deacetylase 1

MacromoleculeName: Histone deacetylase 1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAQTQGTRRK VCYYYDGDVG NYYYGQGHPM KPHRIRMTHN LLLNYGLYRK MEIYRPHKAN AEEMTKYHS DDYIKFLRSI RPDNMSEYSK QMQRFNVGED CPVFDGLFEF CQLSTGGSVA S AVKLNKQQ TDIAVNWAGG LHHAKKSEAS GFCYVNDIVL AILELLKYHQ ...String:
MAQTQGTRRK VCYYYDGDVG NYYYGQGHPM KPHRIRMTHN LLLNYGLYRK MEIYRPHKAN AEEMTKYHS DDYIKFLRSI RPDNMSEYSK QMQRFNVGED CPVFDGLFEF CQLSTGGSVA S AVKLNKQQ TDIAVNWAGG LHHAKKSEAS GFCYVNDIVL AILELLKYHQ RVLYIDIDIH HG DGVEEAF YTTDRVMTVS FHKYGEYFPG TGDLRDIGAG KGKYYAVNYP LRDGIDDESY EAI FKPVMS KVMEMFQPSA VVLQCGSDSL SGDRLGCFNL TIKGHAKCVE FVKSFNLPML MLGG GGYTI RNVARCWTYE TAVALDTEIP NELPYNDYFE YFGPDFKLHI SPSNMTNQNT NEYLE KIKQ RLFENLRMLP HAPGVQMQAI PEDAIPEESG DEDEDDPDKR ISICSSDKRI ACEEEF SDS EEEGEGGRKN SSNFKKAKRV KTEDEKEKDP EEKKEVTEEE KTKEEKPEAK GVKEEVK LA

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Macromolecule #3: Methyl-CpG-binding protein MBD3/GATA zinc finger domain-containin...

MacromoleculeName: Methyl-CpG-binding protein MBD3/GATA zinc finger domain-containing protein 2A fusion
type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MERKRWECPA LPQGWEREEV PRRSGLSAGH RDVFYYSPSG KKFRSKPQLA RYLGGSMDLS TFDFRTGKM LMNKMNKSRQ RVRYDSSNQV KGKPDLNTAL PVRQTASIFK QPVTKITNHP S NKVKSDPQ KAVDQPRQLF WEKKLSGLSA FDIAEELVRT MDLPKGLQGV ...String:
MERKRWECPA LPQGWEREEV PRRSGLSAGH RDVFYYSPSG KKFRSKPQLA RYLGGSMDLS TFDFRTGKM LMNKMNKSRQ RVRYDSSNQV KGKPDLNTAL PVRQTASIFK QPVTKITNHP S NKVKSDPQ KAVDQPRQLF WEKKLSGLSA FDIAEELVRT MDLPKGLQGV GPGCTDETLL SA IASALHT STLPITGQLS AAVEKNPGVW LNTAQPLCKA FMVTDDDIRK QEELVQQVRK RLE EALMAD MLAHVEELAR DGEAPLDKAC AEEEEEEEEE EEEPEPERVS PEERERMIKQ LKEELRLEEA KLVLLKKLRQ SQIQKEATAQ K

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.027 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMHEPES-KOH
75.0 mMSodium ChlorideNaCl
3.0 mMDTT
StainingType: NEGATIVE / Material: Uranyl Acetate
Details: Sample (5 microliters) was applied to a glow-discharged, carbon-coated 400-mesh copper grid (GSCu400CC, ProSciTech). After 2 minutes incubation time, the grid was blotted and washed with ten ...Details: Sample (5 microliters) was applied to a glow-discharged, carbon-coated 400-mesh copper grid (GSCu400CC, ProSciTech). After 2 minutes incubation time, the grid was blotted and washed with ten drops of distilled water, blotting on a filter paper in between washes. The grid was then washed in a drop of 1 % (w/v) uranyl acetate, blotted and subsequently incubated in another drop of 1% (w/v) uranyl acetate solution for 30 seconds, blotted and allowed to air dry at room temperature.
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
DetailsSample was a sample from GraFix purification.

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Electron microscopy

MicroscopeFEI TECNAI 12
Image recordingFilm or detector model: OTHER / Number grids imaged: 1 / Number real images: 325 / Average electron dose: 10.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

Particle selectionNumber selected: 49216
CTF correctionSoftware - Name: RELION (ver. 2.0)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4bkx


Details: We used as a startup model the PDB structure of the MTA1-HDAC1 dimer (PDB 4BKX).
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 29.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 8233
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 1 / Avg.num./class: 8233 / Software - Name: RELION (ver. 2.0)
FSC plot (resolution estimation)

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