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

Basic information

Entry

Database: EMDB / ID: EMD-27557

• Title: Negative stain EM map of an MTA-HDAC-RBBP4 complex
• Map data: Main map of MHR after homogeneous refinement.

Sample

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

• Keywords: Complex / Subcomplex / Deacetylase / Remodeller / Protein Binding / DNA BINDING PROTEIN
• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / negative staining / Resolution: 26.0 Å
• Authors: Jackman MJ / Landsberg MJ / Viswanath S / Arvindekar S / Mackay JP

Funding support

Australia, India, 6 items

Organization	Grant number	Country
National Health and Medical Research Council (NHMRC, Australia)	APP1126357	Australia
Department of Science & Technology (DST, India)	SPG/2020/000475	India
Other government	RTI 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

• Citation: Journal: 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 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

Deposition	Jul 11, 2022	-
Header (metadata) release	Sep 7, 2022	-
Map release	Sep 7, 2022	-
Update	Jan 17, 2024	-
Current status	Jan 17, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_27557.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Main map of MHR after homogeneous refinement.
• Voxel size: X=Y=Z: 2.79 Å

Density

Contour Level	By AUTHOR: 0.179
Minimum - Maximum	-1.1987458 - 5.360952
Average (Standard dev.)	-0.01355195 (±0.1771658)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 250 250 250 Spacing 250 250 250
Cell	A=B=C: 697.5 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_27557_msk_1.map

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

• File: emd_27557_additional_1.map
• Annotation: Input 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.

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

• File: emd_27557_half_map_1.map
• Annotation: Half map B from homogeneous refinement that produced final map of MHR.

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

• File: emd_27557_half_map_2.map
• Annotation: Half map A from homogeneous refinement that produced final map of MHR.

Sample components

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

• Entire: Name: 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

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

• Supramolecule: Name: 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)

Experimental details

Structure determination

• Method: negative staining
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.015 mg/mL

Buffer

pH: 7.5
Component:

Concentration	Formula	Name
10.0 mM	C8H19KN2O5S	HEPES-KOH
75.0 mM	NaCl	sodium chloride
0.3 mM	C4H10O2S2	dithiothreitol

Details: This is the exchange buffer used for both concentrating the sample and also removing sucrose after GraFix.

• Staining: Type: 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 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.
• Grid: Model: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5 / Pretreatment - Type: GLOW DISCHARGE
• Details: The 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.

Electron microscopy

• Microscope: FEI TECNAI 12
• Image recording: Film or detector model: OTHER / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 469 / Average electron dose: 100.0 e/Ų; Details: Images recorded on a DE LC1100 lens coupled CCD detector. Estimated dose.
• Electron beam: Acceleration voltage: 120 kV / Electron source: LAB6
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000

Image processing

• Details: The 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).
• Particle selection: Number 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 model: Type of model: OTHER; Details: The model was created using Ab-initio Reconstruction, as a means to judge the quality of the data.
• Final reconstruction: Number 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
• Initial angle assignment: Type: 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 angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: See above. / Details: See above.
• Final 3D classification: Number 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.