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- EMDB-27557: Negative stain EM map of an MTA-HDAC-RBBP4 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-27557
TitleNegative stain EM map of an MTA-HDAC-RBBP4 complex
Map dataMain map of MHR after homogeneous refinement.
Sample
  • Complex: Low resolution map of the MTA1:HDAC1:RBBP7 complex from HEK Expi293F cells
KeywordsComplex / Subcomplex / Deacetylase / Remodeller / Protein Binding / DNA BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsJackman MJ / Landsberg MJ / Viswanath S / Arvindekar S / Mackay JP
Funding support Australia, India, 6 items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)APP1126357 Australia
Department of Science & Technology (DST, India)SPG/2020/000475 India
Other governmentRTI 4006 India
National Health and Medical Research Council (NHMRC, Australia)APP1012161 Australia
National Health and Medical Research Council (NHMRC, Australia)APP1063301 Australia
National Health and Medical Research Council (NHMRC, Australia)APP1058916 Australia
CitationJournal: Protein Sci / Year: 2022
Title: Molecular architecture of nucleosome remodeling and deacetylase sub-complexes by integrative structure determination.
Authors: Shreyas Arvindekar / Matthew J Jackman / Jason K K Low / Michael J Landsberg / Joel P Mackay / Shruthi Viswanath /
Abstract: The nucleosome remodeling and deacetylase (NuRD) complex is a chromatin-modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information ...The nucleosome remodeling and deacetylase (NuRD) complex is a chromatin-modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information describing the complete NuRD complex is available and a detailed understanding of its mechanism is therefore lacking. Drawing on information from SEC-MALLS, DIA-MS, XLMS, negative-stain EM, X-ray crystallography, NMR spectroscopy, secondary structure predictions, and homology models, we applied Bayesian integrative structure determination to investigate the molecular architecture of three NuRD sub-complexes: MTA1-HDAC1-RBBP4, MTA1 -HDAC1-MBD3 , and MTA1-HDAC1-RBBP4-MBD3-GATAD2A [nucleosome deacetylase (NuDe)]. The integrative structures were corroborated by examining independent crosslinks, cryo-EM maps, biochemical assays, known cancer-associated mutations, and structure predictions from AlphaFold. The robustness of the models was assessed by jack-knifing. Localization of the full-length MBD3, which connects the deacetylase and chromatin remodeling modules in NuRD, has not previously been possible; our models indicate two different locations for MBD3, suggesting a mechanism by which MBD3 in the presence of GATAD2A asymmetrically bridges the two modules in NuRD. Further, our models uncovered three previously unrecognized subunit interfaces in NuDe: HDAC1 -MTA1 , MTA1 -MBD3 , and HDAC1 -MBD3 . Our approach also allowed us to localize regions of unknown structure, such as HDAC1 and MBD3 , thereby resulting in the most complete and robustly cross-validated structural characterization of these NuRD sub-complexes so far.
History
DepositionJul 11, 2022-
Header (metadata) releaseSep 7, 2022-
Map releaseSep 7, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27557.png

Downloads & links

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Map

FileDownload / File: emd_27557.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map of MHR after homogeneous refinement.
Voxel sizeX=Y=Z: 2.79 Å
Density
Contour LevelBy AUTHOR: 0.179
Minimum - Maximum-1.1987458 - 5.360952
Average (Standard dev.)-0.01355195 (±0.1771658)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 697.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27557_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Input used in the IMP for the generation...

Fileemd_27557_additional_1.map
AnnotationInput used in the IMP for the generation of the predicted MHR model. It was derived from an independent processing run using the same dataset as the model used to generate the final map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B from homogeneous refinement that produced...

Fileemd_27557_half_map_1.map
AnnotationHalf map B from homogeneous refinement that produced final map of MHR.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A from homogeneous refinement that produced...

Fileemd_27557_half_map_2.map
AnnotationHalf map A from homogeneous refinement that produced final map of MHR.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Low resolution map of the MTA1:HDAC1:RBBP7 complex from HEK Expi2...

EntireName: Low resolution map of the MTA1:HDAC1:RBBP7 complex from HEK Expi293F cells
Components
  • Complex: Low resolution map of the MTA1:HDAC1:RBBP7 complex from HEK Expi293F cells

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Supramolecule #1: Low resolution map of the MTA1:HDAC1:RBBP7 complex from HEK Expi2...

SupramoleculeName: Low resolution map of the MTA1:HDAC1:RBBP7 complex from HEK Expi293F cells
type: complex / ID: 1 / Parent: 0
Details: Subcomplex that was generated via combination of recombinant proteins pulled down via FLAG tags from HEK Expi293F cells
Source (natural)Organism: Homo sapiens (human)

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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

Concentration0.015 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMC8H19KN2O5SHEPES-KOH
75.0 mMNaClSodium chloridesodium chloride
0.3 mMC4H10O2S2dithiothreitol

Details: This is the exchange buffer used for both concentrating the sample and also removing sucrose after GraFix.
StainingType: NEGATIVE / Material: Uranyl Acetate
Details: 5 uL of sample was applied to the grid and incubated for 2 minutes. The grid was then blotted and washed with 10 drops of distilled water, with liquid blotted off between each drop. The grid ...Details: 5 uL of sample was applied to the grid and incubated for 2 minutes. The grid was then blotted and washed with 10 drops of distilled water, with liquid blotted off between each drop. The grid was then pre-stained with a drop 1% Uranyl Acetate, blotted, then stained with a drop of 1% Uranyl Acetate for 30 seconds. It was then blotted again and left to air dry at room temperature.
GridModel: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5 / Pretreatment - Type: GLOW DISCHARGE
DetailsThe sample was in high abundance, and appears to be well dispersed. Some aggregation is clear, and the particles upon closer inspection (such as 2D classification) are heterogeneous. This is likely due to the flexibility of the complex.

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 469 / Average electron dose: 100.0 e/Å2
Details: Images recorded on a DE LC1100 lens coupled CCD detector. Estimated dose.

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Image processing

Particle selectionNumber selected: 914180
Details: Use of template from a previous reconstruction run of MHR. This was used for template picking, and the chosen particles were then extracted and underwent 2D Classification.
Startup modelType of model: OTHER
Details: The model was created using Ab-initio Reconstruction, as a means to judge the quality of the data.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Software - details: This occurs as above during Ab-initio reconstruction.
Details: This is used in Ab-initio Reconstruction.
Final 3D classificationNumber classes: 50 / Avg.num./class: 1546 / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: See above.
Details: 2D classification was used before Ab-initio Reconstruction as a cleanup step.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: See above. / Details: See above.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1)
Software - details: Homogeneous refinement (the newest version) was used.
Details: Homogeneous refinement uses a Gold Standard approach to determine the resolution of the map.
Number images used: 45954
DetailsThe images selected underwent CTF estimation, with which blob picking was used to select particles with a maximum diameter of about 250 Angstroms (based on information we previously had about the particle size).
FSC plot (resolution estimation)

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