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- EMDB-12704: Cooperation between the intrinsically disordered and ordered regi... -

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Basic information

Entry
Database: EMDB / ID: EMD-12704
TitleCooperation between the intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly
Map dataCryosparc sharpened electron density map
Sample
  • Organelle or cellular component: Structure of elongation factor Spt6
    • Protein or peptide: Transcription elongation factor SPT6
Function / homology
Function and homology information


carbon catabolite repression of transcription from RNA polymerase II promoter by glucose / regulation of transcriptional start site selection at RNA polymerase II promoter / transcription antitermination factor activity, DNA binding / regulation of mRNA 3'-end processing / nucleosome organization / transcription elongation-coupled chromatin remodeling / poly(A)+ mRNA export from nucleus / nucleosome binding / transcription elongation factor complex / transcription antitermination ...carbon catabolite repression of transcription from RNA polymerase II promoter by glucose / regulation of transcriptional start site selection at RNA polymerase II promoter / transcription antitermination factor activity, DNA binding / regulation of mRNA 3'-end processing / nucleosome organization / transcription elongation-coupled chromatin remodeling / poly(A)+ mRNA export from nucleus / nucleosome binding / transcription elongation factor complex / transcription antitermination / transcription elongation by RNA polymerase II / positive regulation of transcription elongation by RNA polymerase II / euchromatin / nucleosome assembly / chromatin organization / histone binding / chromatin remodeling / regulation of transcription by RNA polymerase II / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / mitochondrion / DNA binding / nucleus
Similarity search - Function
: / Spt6, S1/OB domain / YqgF/RNase H-like domain / Likely ribonuclease with RNase H fold. / Spt6 acidic, N-terminal domain / Helix-turn-helix DNA-binding domain of Spt6 / Transcription elongation factor Spt6, YqgF domain / Transcription elongation factor Spt6, helix-hairpin-helix motif / Spt6, SH2 domain, C terminus / Acidic N-terminal SPT6 ...: / Spt6, S1/OB domain / YqgF/RNase H-like domain / Likely ribonuclease with RNase H fold. / Spt6 acidic, N-terminal domain / Helix-turn-helix DNA-binding domain of Spt6 / Transcription elongation factor Spt6, YqgF domain / Transcription elongation factor Spt6, helix-hairpin-helix motif / Spt6, SH2 domain, C terminus / Acidic N-terminal SPT6 / Helix-hairpin-helix motif / Holliday-junction resolvase-like of SPT6 / Helix-turn-helix DNA-binding domain of SPT6 / Tex-like protein, HTH domain superfamily / Tex-like domain superfamily / Spt6, Death-like domain / Transcription elongation factor Spt6 / Spt6, SH2 domain, N terminus / Spt6, SH2 domain / SH2 domain / YqgF/RNase H-like domain superfamily / RuvA domain 2-like / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / SH2 domain superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
Transcription elongation factor SPT6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsKasiliauskaite A / Kubicek K / Klumpler T / Zanova M / Zapletal D / Novacek J / Stefl R
Funding support Czech Republic, 6 items
OrganizationGrant numberCountry
Ministry of Education, Youth and Sports of the Czech RepublicLM20115043 Czech Republic
Ministry of Education, Youth and Sports of the Czech RepublicLM20115070 Czech Republic
Ministry of Education, Youth and Sports of the Czech RepublicLM20115042 Czech Republic
European Research Council (ERC)649030 Czech Republic
Ministry of Education, Youth and Sports of the Czech RepublicLQ1601 Czech Republic
Czech Science FoundationGA18-11397S Czech Republic
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly.
Authors: Aiste Kasiliauskaite / Karel Kubicek / Tomas Klumpler / Martina Zanova / David Zapletal / Eliska Koutna / Jiri Novacek / Richard Stefl /
Abstract: Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The ...Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The precise mechanism of nucleosome reassembly mediated by Spt6 remains unclear. In this study, we used a hybrid approach combining cryo-electron microscopy and small-angle X-ray scattering to visualize the architecture of Spt6 from Saccharomyces cerevisiae. The reconstructed overall architecture of Spt6 reveals not only the core of Spt6, but also its flexible N- and C-termini, which are critical for Spt6's function. We found that the acidic N-terminal region of Spt6 prevents the binding of Spt6 not only to the Pol II CTD and Pol II CTD-linker, but also to pre-formed intact nucleosomes and nucleosomal DNA. The N-terminal region of Spt6 self-associates with the tSH2 domain and the core of Spt6 and thus controls binding to Pol II and nucleosomes. Furthermore, we found that Spt6 promotes the assembly of nucleosomes in vitro. These data indicate that the cooperation between the intrinsically disordered and structured regions of Spt6 regulates nucleosome and Pol II CTD binding, and also nucleosome assembly.
History
DepositionApr 1, 2021-
Header (metadata) releaseApr 13, 2022-
Map releaseApr 13, 2022-
UpdateJun 15, 2022-
Current statusJun 15, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_12704.png

Downloads & links

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Map

FileDownload / File: emd_12704.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryosparc sharpened electron density map
Voxel sizeX=Y=Z: 0.828 Å
Density
Contour LevelBy AUTHOR: 0.164
Minimum - Maximum-0.46966928 - 0.6867795
Average (Standard dev.)-0.0013449175 (±0.01961691)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_12704_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12704_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of elongation factor Spt6

EntireName: Structure of elongation factor Spt6
Components
  • Organelle or cellular component: Structure of elongation factor Spt6
    • Protein or peptide: Transcription elongation factor SPT6

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Supramolecule #1: Structure of elongation factor Spt6

SupramoleculeName: Structure of elongation factor Spt6 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Recombinant expressionOrganism: Escherichia coli B (bacteria)

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Macromolecule #1: Transcription elongation factor SPT6

MacromoleculeName: Transcription elongation factor SPT6 / type: protein_or_peptide / ID: 1
Details: the sequence stretches of the sample that do not make part of the coordinates are flexible regions that were not observed in the cryoEM map
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 134.923031 KDa
Recombinant expressionOrganism: Escherichia coli B (bacteria)
SequenceString: GIDPFTDIYD LEDLKKNLMT EGDMKIRKTD IPERYQELRA GITDYGNMSS EDQELERNWI AEKISVDKNF DANYDLTEFK EAIGNAIKF ITKENLEVPF IYAYRRNYIS SREKDGFLLT EDDLWDIVSL DIEFHSLVNK KDYVQRFYAE LHIDDPIVTE Y FKNQNTAS ...String:
GIDPFTDIYD LEDLKKNLMT EGDMKIRKTD IPERYQELRA GITDYGNMSS EDQELERNWI AEKISVDKNF DANYDLTEFK EAIGNAIKF ITKENLEVPF IYAYRRNYIS SREKDGFLLT EDDLWDIVSL DIEFHSLVNK KDYVQRFYAE LHIDDPIVTE Y FKNQNTAS IAELNSLQDI YDYLEFKYAN EINEMFINHT GKTGKKHLKN SSYEKFKASP LYQAVSDIGI SAEDVGENIS SQ HQIHPPV DHPSSKPVEV IESILNANSG DLQVFTSNTK LAIDTVQKYY SLELSKNTKI REKVRSDFSK YYLADVVLTA KGK KEIQKG SLYEDIKYAI NRTPMHFRRD PDVFLKMVEA ESLNLLSVKL HMSSQAQYIE HLFQIALETT NTSDIAIEWN NFRK LAFNQ AMDKIFQDIS QEVKDNLTKN CQKLVAKTVR HKFMTKLDQA PFIPNVRDPK IPKILSLTCG QGRFGADAII AVYVN RKGD FIRDYKIVDN PFDKTNPEKF EDTLDNIIQS CQPNAIGING PNPKTQKFYK RLQEVLHKKQ IVDSRGHTIP IIYVED EVA IRYQNSERAA QEFPNKPPLV KYCIALARYM HSPLLEYANL TSEEVRSLSI HPHQNLLSSE QLSWALETAF VDIVNLV SV EVNKATDNNY YASALKYISG FGKRKAIDFL QSLQRLNEPL LARQQLITHN ILHKTIFMNS AGFLYISWNE KRQKYEDL E HDQLDSTRIH PEDYHLATKV AADALEYDPD TIAEKEEQGT MSEFIELLRE DPDRRAKLES LNLESYAEEL EKNTGLRKL NNLNTIVLEL LDGFEELRND FHPLQGDEIF QSLTGESEKT FFKGSIIPVR VERFWHNDII CTTNSEVECV VNAQRHAGAQ LRRPANEIY EIGKTYPAKV IYIDYANITA EVSLLDHDVK QQYVPISYSK DPSIWDLKQE LEDAEEERKL MMAEARAKRT H RVINHPYY FPFNGRQAED YLRSKERGEF VIRQSSRGDD HLVITWKLDK DLFQHIDIQE LEKENPLALG KVLIVDNQKY ND LDQIIVE YLQNKVRLLN EMTSSEKFKS GTKKDVVKFI EDYSRVNPNK SVYYFSLNHD NPGWFYLMFK INANSKLYTW NVK LTNTGY FLVNYNYPSV IQLCNGFKTL LKSNSSKNRM NNYR

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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

Concentration0.15 mg/mL
BufferpH: 7.5 / Component - Concentration: 20.0 mM / Component - Formula: HEPES / Component - Name: HEPES
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.007 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Detailshomogeneous monodisperse sample

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3840 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 13807 / Average exposure time: 5.0 sec. / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2995892
CTF correctionSoftware - Name: cryoSPARC (ver. 3.1)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3psi


Details: Initial PDB coordinates of the Spt6 structure comprising residues 298-1248 were produced from PDBs of a crystal structure of Spt6, namely 3PSI and 3PSF. The PDB and the cryoSPARC sharpened ...Details: Initial PDB coordinates of the Spt6 structure comprising residues 298-1248 were produced from PDBs of a crystal structure of Spt6, namely 3PSI and 3PSF. The PDB and the cryoSPARC sharpened electron density map were imported into the program Coot and the tool 'Real Space Refine Zone' was used to achiev e optimal fit of the PDB coordinates within the map. Low-resolution regions of the map (regions corresponding to residues: 1226-1243 and the S1-domain) were also excluded from the PDB and were docked with a rigid body strategy using Phenix once the fit of the well-resolved regions exhibited no clashes and deviations.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final 3D classificationNumber classes: 313 / Avg.num./class: 2296 / Software - Name: cryoSPARC (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final reconstructionNumber classes used: 313 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 718639

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Atomic model buiding 1

Initial model
PDB IDChain

3psi

chain_id: A, residue_range: 1127-1219

3psf

chain_id: A, residue_range: 298-1248
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficients
Output model

PDB-7o3d:
Cooperation between the intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly

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