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- EMDB-3517: Structural reorganization of the chromatin remodeling enzyme Chd1... -

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Basic information

Entry
Database: EMDB / ID: EMD-3517
TitleStructural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes.
Map dataS.Cerevisiae Chd1 bound to 601-Nucleosome
Sample
  • Complex: Chd1-Nucleosome complex
    • Complex: Chd1
    • Complex: Nucleosome
    • Other: Chromodomain Helicase DNA binding protein
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Xenopus laevis (African clawed frog)
Methodsingle particle reconstruction / cryo EM / Resolution: 20.0 Å
AuthorsSundaramoorthy R / Owen-Hughes T
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Elife / Year: 2017
Title: Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes.
Authors: Ramasubramanian Sundaramoorthy / Amanda L Hughes / Vijender Singh / Nicola Wiechens / Daniel P Ryan / Hassane El-Mkami / Maxim Petoukhov / Dmitri I Svergun / Barbara Treutlein / Salina Quack ...Authors: Ramasubramanian Sundaramoorthy / Amanda L Hughes / Vijender Singh / Nicola Wiechens / Daniel P Ryan / Hassane El-Mkami / Maxim Petoukhov / Dmitri I Svergun / Barbara Treutlein / Salina Quack / Monika Fischer / Jens Michaelis / Bettina Böttcher / David G Norman / Tom Owen-Hughes /
Abstract: The yeast Chd1 protein acts to position nucleosomes across genomes. Here, we model the structure of the Chd1 protein in solution and when bound to nucleosomes. In the apo state, the DNA-binding ...The yeast Chd1 protein acts to position nucleosomes across genomes. Here, we model the structure of the Chd1 protein in solution and when bound to nucleosomes. In the apo state, the DNA-binding domain contacts the edge of the nucleosome while in the presence of the non-hydrolyzable ATP analog, ADP-beryllium fluoride, we observe additional interactions between the ATPase domain and the adjacent DNA gyre 1.5 helical turns from the dyad axis of symmetry. Binding in this conformation involves unravelling the outer turn of nucleosomal DNA and requires substantial reorientation of the DNA-binding domain with respect to the ATPase domains. The orientation of the DNA-binding domain is mediated by sequences in the N-terminus and mutations to this part of the protein have positive and negative effects on Chd1 activity. These observations indicate that the unfavorable alignment of C-terminal DNA-binding region in solution contributes to an auto-inhibited state.
History
DepositionNov 25, 2016-
Header (metadata) releaseDec 28, 2016-
Map releaseApr 5, 2017-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0145
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0145
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3517.png

Downloads & links

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Map

FileDownload / File: emd_3517.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationS.Cerevisiae Chd1 bound to 601-Nucleosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.58 Å/pix.
x 200 pix.
= 316. Å		1.58 Å/pix.
x 200 pix.
= 316. Å		1.58 Å/pix.
x 200 pix.
= 316. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.58 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0145 / Movie #1: 0.0145
Minimum - Maximum-0.021911494 - 0.14400715
Average (Standard dev.)0.0008408567 (±0.0077083986)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 316.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.581.581.58
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z316.000316.000316.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0220.1440.001

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Supplemental data

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Sample components

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Entire : Chd1-Nucleosome complex

EntireName: Chd1-Nucleosome complex
Components
  • Complex: Chd1-Nucleosome complex
    • Complex: Chd1
    • Complex: Nucleosome
    • Other: Chromodomain Helicase DNA binding protein

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Supramolecule #1: Chd1-Nucleosome complex

SupramoleculeName: Chd1-Nucleosome complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: S.Cerevisiae chromatin remodelling enzyme in complex with 601-nucleosome
Molecular weightTheoretical: 400 KDa

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Supramolecule #2: Chd1

SupramoleculeName: Chd1 / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Nucleosome

SupramoleculeName: Nucleosome / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Chromodomain Helicase DNA binding protein

MacromoleculeName: Chromodomain Helicase DNA binding protein / type: other / ID: 1 / Details: Protein / Classification: other
SequenceString: MAAKDISTEV LQNPELYGLR RSHRAAAHQQ NYFNDSDDED DEDNIKQSRR KRMTTIEDDE DEFEDEEGE EDSGEDEDEE DFEEDDDYYG SPIKQNRSKP KSRTKSKSKS KPKSQSEKQS T VKIPTRFS NRQNKTVNYN IDYSDDDLLE SEDDYGSEEA LSEENVHEAS ...String:
MAAKDISTEV LQNPELYGLR RSHRAAAHQQ NYFNDSDDED DEDNIKQSRR KRMTTIEDDE DEFEDEEGE EDSGEDEDEE DFEEDDDYYG SPIKQNRSKP KSRTKSKSKS KPKSQSEKQS T VKIPTRFS NRQNKTVNYN IDYSDDDLLE SEDDYGSEEA LSEENVHEAS ANPQPEDFHG ID IVINHRL KTSLEEGKVL EKTVPDLNNC KENYEFLIKW TDESHLHNTW ETYESIGQVR GLK RLDNYC KQFIIEDQQV RLDPYVTAED IEIMDMERER RLDEFEEFHV PERIIDSQRA SLED GTSQL QYLVKWRRLN YDEATWENAT DIVKLAPEQV KHFQNRENSK ILPQYSSNYT SQRPR FEKL SVQPPFIKGG ELRDFQLTGI NWMAFLWSKG DNGILADEMG LGKTVQTVAF ISWLIF ARR QNGPHIIVVP LSTMPAWLDT FEKWAPDLNC ICYMGNQKSR DTIREYEFYT NPRAKGK KT MKFNVLLTTY EYILKDRAEL GSIKWQFMAV DEAHRLKNAE SSLYESLNSF KVANRMLI T GTPLQNNIKE LAALVNFLMP GRFTIDQEID FENQDEEQEE YIHDLHRRIQ PFILRRLKK DVEKSLPSKT ERILRVELSD VQTEYYKNIL TKNYSALTAG AKGGHFSLLN IMNELKKASN HPYLFDNAE ERVLQKFGDG KMTRENVLRG LIMSSGKMVL LDQLLTRLKK DGHRVLIFSQ M VRMLDILG DYLSIKGINF QRLDGTVPSA QRRISIDHFN SPDSNDFVFL LSTRAGGLGI NL MTADTVV IFDSDWNPQA DLQAMARAHR IGQKNHVMVY RLVSKDTVEE EVLERARKKM ILE YAIISL GVTDGNKYTK KNEPNAGELS AILKFGAGNM FTATDNQKKL EDLNLDDVLN HAED HVTTP DLGESHLGGE EFLKQFEVTD YKADIDWDDI IPEEELKKLQ DEEQKRKDEE YVKEQ LEMM NRRDNALKKI KNSVNGDGTA ANSDSDDDST SRSSRRRARA NDMDSIGESE VRALYK AIL KFGNLKEILD ELIADGTLPV KSFEKYGETY DEMMEAAKDC VHEEEKNRKE ILEKLEK HA TAYRAKLKSG EIKAENQPKD NPLTRLSLKK REKKAVLFNF KGVKSLNAES LLSRVEDL K YLKNLINSNY KDDPLKFSLG NNTPKPVQNW SSNWTKEEDE KLLIGVFKYG YGSWTQIRD DPFLGITDKI FLNEVHNPVA KKSASSSDTT PTPSKKGKGI TGSSKKVPGA IHLGRRVDYL LSFLRGGLN TKSPSADIGS KKLPTGPSKK RQRKPANHSK SMTPEI

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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

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMTristris(hydroxymethyl)aminomethane
50.0 mMNaClSodium Chloride

Details: Solutions were made fresh.
GridModel: Quantifoil / Material: COPPER/RHODIUM / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Grids are double side blotted with 2sec blotting time and blotting force 10.
DetailsThis sample was monodisperse. One Chd1 molecule bound to one Nucleosome.

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureMin: 70.0 K / Max: 70.0 K
DetailsPreliminary grid screen was done manually and then data collection was performed automatically using EMTools-EMMenu
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 15.6 µm / Number grids imaged: 1 / Number real images: 140 / Average exposure time: 2.0 sec. / Average electron dose: 22.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 68000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 68000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

DetailsImages were manually inspected for any contamination or damage.
Particle selectionNumber selected: 47000
Details: Initial template for autopicking is made from manually picked particles. Then RELION-1.3 autopick routine was used for autopicking
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1kx5


Details: Nucleosome structure 1KX5 was converted into map of pixel size 1.58, low pass filtered and used as initial model.
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 36324
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: PROJECTION MATCHING / Projection matching processing - Merit function: CC
Projection matching processing - Angular sampling: 7.5 degrees
Software - Name: RELION (ver. 1.3)
Final 3D classificationNumber classes: 2 / Avg.num./class: 17000 / Software - Name: RELION (ver. 1.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1kx5

chain_id: A, B, C, D, E, F, G, H, I, J

3mwy

chain_id: A, residue_range: 175-932

2xb0

chain_id: X, residue_range: 1010-1274
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 2800 / Target criteria: Cross-correlation coefficient

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