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- EMDB-20393: CryoEM structure of wild type mediator with MED25-FLAG -

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Basic information

Entry
Database: EMDB / ID: EMD-20393
TitleCryoEM structure of wild type mediator with MED25-FLAG
Map dataem-volume_P1
Sample
  • Complex: 30 proteins complex
    • Other: protein complex
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.49 Å
AuthorsAsturias F / Zhao H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01-67167 United States
CitationJournal: Cell / Year: 2019
Title: A Pliable Mediator Acts as a Functional Rather Than an Architectural Bridge between Promoters and Enhancers.
Authors: Laila El Khattabi / Haiyan Zhao / Jens Kalchschmidt / Natalie Young / Seolkyoung Jung / Peter Van Blerkom / Philippe Kieffer-Kwon / Kyong-Rim Kieffer-Kwon / Solji Park / Xiang Wang / Jordan ...Authors: Laila El Khattabi / Haiyan Zhao / Jens Kalchschmidt / Natalie Young / Seolkyoung Jung / Peter Van Blerkom / Philippe Kieffer-Kwon / Kyong-Rim Kieffer-Kwon / Solji Park / Xiang Wang / Jordan Krebs / Subhash Tripathi / Noboru Sakabe / Débora R Sobreira / Su-Chen Huang / Suhas S P Rao / Nathanael Pruett / Daniel Chauss / Erica Sadler / Andrea Lopez / Marcelo A Nóbrega / Erez Lieberman Aiden / Francisco J Asturias / Rafael Casellas /
Abstract: While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron ...While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.
History
DepositionJul 1, 2019-
Header (metadata) releaseAug 7, 2019-
Map releaseAug 21, 2019-
UpdateMar 4, 2020-
Current statusMar 4, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20393.png

Downloads & links

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Map

FileDownload / File: emd_20393.map.gz / Format: CCP4 / Size: 669.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationem-volume_P1
Voxel sizeX=Y=Z: 1.11 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.5366784 - 1.4533664
Average (Standard dev.)-0.00042138158 (±0.034071453)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions560560560
Spacing560560560
CellA=B=C: 621.60004 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.111.111.11
M x/y/z560560560
origin x/y/z0.0000.0000.000
length x/y/z621.600621.600621.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS560560560
D min/max/mean-0.5371.453-0.000

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Supplemental data

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Sample components

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Entire : 30 proteins complex

EntireName: 30 proteins complex
Components
  • Complex: 30 proteins complex
    • Other: protein complex

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Supramolecule #1: 30 proteins complex

SupramoleculeName: 30 proteins complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Mus musculus (house mouse) / Recombinant cell: CH12 B cells
Molecular weightTheoretical: 1.0 MDa

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Macromolecule #1: protein complex

MacromoleculeName: protein complex / type: other / ID: 1 / Classification: other
Source (natural)Organism: Mus musculus (house mouse)
SequenceString: MAPVQLDNHQ LIPPGGGGGS SGGGGSSSGS ASAPAPPPPA AAVAAAAAAA ASPGYRLSTL IEFLLHRAY SELMVLTDLL PRKSDVERKI EIVQFASRTR QLFVRLLALV KWANDAGKVE K CAMISSFL DQQAILFVDT ADRLASLARD ALVHARLPSF AIPYAIDVLT ...String:
MAPVQLDNHQ LIPPGGGGGS SGGGGSSSGS ASAPAPPPPA AAVAAAAAAA ASPGYRLSTL IEFLLHRAY SELMVLTDLL PRKSDVERKI EIVQFASRTR QLFVRLLALV KWANDAGKVE K CAMISSFL DQQAILFVDT ADRLASLARD ALVHARLPSF AIPYAIDVLT TGSYPRLPTC IR DKIIPPD PITKIEKQAT LHQLNQILRH RLVTTDLPPQ LANLTVANGR VKFRVEGEFE ATL TVMGDD PEVPWRLLKL EILVEDKETG DGRALVHSMQ IDFIHQLVQS RLFADEKPLQ DMYN CLHCF CLSLQLEVLH SQTLMLIRER WGDLVQVERY HAGKSLSLSV WNQQVLGRKT GTASV HKVT IKIDENDVSK PLQIFHDPPL PASDSKLVER AMKIDHLSIE KLLIDSVHAR AHQRLQ ELK AILRSFNANE SSSIETALPA LIVPILEPCG NSECLHIFVD LHSGMFQLML YGLDPAT LE DMEKSLNDDM KRIIPWIQQL KFWLGQQRCK QSIKHLPTIT TETLQLANYS THPIGSLS K NKLFIKLTRL PQYYIVVEML EVPNKPTQLS YNYYFMSVST ADREDSPVMA LLLQQFKDN IQDLMSYTKT GKQTRTGTKH KLSDDPCPID SKKAKRSGEM CAFNKVLAHF VAMCDTNMPF VGLRLELSN LEIPHQGVQV EGDGFNHAIR LLKIPPCKGI SEETQKALDR SLLDCTFRLQ G RNNRTWVA ELVFANCPLN GTSTREQGPS RHVYLTYENL LSEPVGGRKV VEMFLNDWSS IA RLYECVL EFARSLPEIP AHLNIFSEVR VYNYRKLILC YGTTKGSSIS IQWNSIHQKF HIA LGTVGP NSGCSNCHNT ILHQLQEMFN KTPNVVQLLQ VLFDTQAPLN AINKLPTVPM LGLT QRTNT AYQCFSILPQ SSTHIRLAFR NMYCIDIYCR SRGVVAIRDG AYSLFDNSKL VEGFY PAPG LKTFLNMFVD SNQDARRRSV NEDDNPPSPI GGDMMDSLIS QLQPPQQQPF PKQPGT SGA YPLTSPPTSY HSTVNQSPSM MHTQSPGNLH AASSPSGALR APSPASFVPT PPPSSHG IS IGPGASFASP HGTLDPSSPY TMVSPSGRAG NWPGSPQVSG PSPATRLPGM SPANPSLH S PVPDVSHSPR AGTSSQTMPT NMPPPRKLPQ RSWAASIPTI LTHSALNILL LPSPTPGLV PGLAGSYLCS PLERFLGSVI MRRHLQRIIQ QETLQLINSN EPGVIMFKTD ALKCRVALSP KTNQTLQLK VTPENAGQWK PDELQVLEKF FETRVAGPPF KANTLIAFTK LLGAPTHILR D CVHIMKLE LFPDQATQLK WNVQFCLTIP PSAPPIAPPG TPAVVLKSKM LFFLQLTQKT SV PPQEPVS IIVPIIYDMA SGTTQQADIP RQQNSSVAAP MMVSNILKRF AEMNPPRQGE CTI FAAVRD LMANLTLPPG GRP

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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

Concentration0.5 mg/mL
BufferpH: 7.9 / Component - Concentration: 50.0 mM / Component - Formula: C8H18N2O4S / Component - Name: HEPES
GridMaterial: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 3.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 273 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 1.2 µm / Calibrated magnification: 22000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 22000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Frames/image: 32-40 / Number grids imaged: 1 / Number real images: 3854 / Average exposure time: 3.0 sec. / Average electron dose: 53.9 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 136952
CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 200 / Avg.num./class: 700
Final angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 136952
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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