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- PDB-5xzc: Cryo-EM structure of p300-p53 protein complex -

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Basic information

Entry
Database: PDB / ID: 5xzc
TitleCryo-EM structure of p300-p53 protein complex
Components
  • Cellular tumor antigen p53P53
  • Histone acetyltransferase p300
KeywordsTRANSCRIPTION / transcription factor / autoacetylation / allosteric interaction / catalytically active form
Function / homology
Function and homology information


peptide butyryltransferase activity / protein propionyltransferase activity / behavioral defense response / peptidyl-lysine crotonylation / peptidyl-lysine butyrylation / histone crotonyltransferase activity / histone H2B acetylation / histone butyryltransferase activity / peptidyl-lysine propionylation / positive regulation of RNA polymerase II regulatory region sequence-specific DNA binding ...peptide butyryltransferase activity / protein propionyltransferase activity / behavioral defense response / peptidyl-lysine crotonylation / peptidyl-lysine butyrylation / histone crotonyltransferase activity / histone H2B acetylation / histone butyryltransferase activity / peptidyl-lysine propionylation / positive regulation of RNA polymerase II regulatory region sequence-specific DNA binding / swimming / regulation of tubulin deacetylation / thigmotaxis / positive regulation of transcription from RNA polymerase II promoter involved in unfolded protein response / lysine N-acetyltransferase activity, acting on acetyl phosphate as donor / peptidyl-lysine acetylation / STAT family protein binding / peptide-lysine-N-acetyltransferase activity / regulation of androgen receptor signaling pathway / positive regulation by host of viral transcription / internal peptidyl-lysine acetylation / positive regulation of gene expression, epigenetic / N-terminal peptidyl-lysine acetylation / internal protein amino acid acetylation / response to vitamin B3 / signal transduction by p53 class mediator / regulation of fibroblast apoptotic process / positive regulation of programmed necrotic cell death / regulation of intrinsic apoptotic signaling pathway by p53 class mediator / negative regulation of glucose catabolic process to lactate via pyruvate / intrinsic apoptotic signaling pathway in response to hypoxia / oxidative stress-induced premature senescence / core promoter sequence-specific DNA binding / regulation of transcription from RNA polymerase II promoter in response to DNA damage / oligodendrocyte apoptotic process / negative regulation of mitophagy / negative regulation of helicase activity / regulation of hydrogen peroxide-induced cell death / DNA strand renaturation / positive regulation of thymocyte apoptotic process / regulation of tissue remodeling / positive regulation of cell aging / megakaryocyte development / bone marrow development / regulation of mitochondrial membrane permeability involved in apoptotic process / circadian behavior / positive regulation of mitochondrial membrane permeability / face morphogenesis / negative regulation of neuroblast proliferation / positive regulation of transcription from RNA polymerase II promoter in response to stress / transferase activity, transferring acyl groups / histone deacetylase regulator activity / positive regulation of transcription of Notch receptor target / transcription cofactor binding / chromatin assembly / regulation of DNA damage response, signal transduction by p53 class mediator / T cell lineage commitment / positive regulation of execution phase of apoptosis / positive regulation of transcription from RNA polymerase II promoter in response to endoplasmic reticulum stress / regulation of cell cycle G2/M phase transition / mRNA transcription / regulation of cellular senescence / pre-mRNA intronic binding / mitotic cell cycle arrest / fat cell differentiation / TFIID-class transcription factor complex binding / MDM2/MDM4 family protein binding / positive regulation of histone deacetylation / macrophage derived foam cell differentiation / negative regulation of DNA replication / mitochondrial DNA repair / B cell lineage commitment / platelet formation / hematopoietic stem cell differentiation / entrainment of circadian clock by photoperiod / rRNA transcription / beta-catenin-TCF complex assembly / positive regulation of transcription from RNA polymerase II promoter in response to hypoxia / positive regulation of cardiac muscle cell apoptotic process / positive regulation of protein oligomerization / positive regulation of leukocyte migration / ER overload response / protein acetylation / positive regulation of transforming growth factor beta receptor signaling pathway / response to UV-B / cellular response to actinomycin D / negative regulation of protein-containing complex assembly / intrinsic apoptotic signaling pathway by p53 class mediator / T cell proliferation involved in immune response / cardiac septum morphogenesis / acetyltransferase activity / RNA polymerase II preinitiation complex assembly / positive regulation of Notch signaling pathway / histone H4 acetylation / DNA damage response, signal transduction by p53 class mediator / response to metal ion / necroptotic process / response to salt stress / embryonic organ development / somitogenesis
P53 DNA-binding domain / Nuclear receptor coactivator, CREB-bp-like, interlocking / Zinc finger, TAZ-type / Zinc finger, ZZ-type / Bromodomain / p53 tumour suppressor family / Coactivator CBP, KIX domain / p53-like transcription factor, DNA-binding / Nuclear receptor coactivator, interlocking / CREB-binding protein/p300, atypical RING domain ...P53 DNA-binding domain / Nuclear receptor coactivator, CREB-bp-like, interlocking / Zinc finger, TAZ-type / Zinc finger, ZZ-type / Bromodomain / p53 tumour suppressor family / Coactivator CBP, KIX domain / p53-like transcription factor, DNA-binding / Nuclear receptor coactivator, interlocking / CREB-binding protein/p300, atypical RING domain / p53, DNA-binding domain / p53/RUNT-type transcription factor, DNA-binding domain superfamily / Zinc finger, RING/FYVE/PHD-type / Histone acetyltransferase Rtt109/CBP / p53 transactivation domain / p53, tetramerisation domain / Bromodomain, conserved site / Nuclear receptor coactivator, CREB-bp-like, interlocking domain superfamily / CBP/p300-type histone acetyltransferase domain / Domain of Unknown Function (DUF902) / Bromodomain / Cellular tumor antigen p53, transactivation domain 2 / CBP/p300, atypical RING domain superfamily / Histone acetylation protein / p53-like tetramerisation domain superfamily / Coactivator CBP, KIX domain superfamily / Bromodomain-like superfamily / TAZ domain superfamily
Cellular tumor antigen p53 / Histone acetyltransferase p300
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 10.7 Å
AuthorsGhosh, R. / Roy, S. / Sengupta, J.
CitationJournal: Biochemistry / Year: 2019
Title: Tumor Suppressor p53-Mediated Structural Reorganization of the Transcriptional Coactivator p300.
Authors: Raka Ghosh / Stephanie Kaypee / Manidip Shasmal / Tapas K Kundu / Siddhartha Roy / Jayati Sengupta /
Abstract: Transcriptional coactivator p300, a critical player in eukaryotic gene regulation, primarily functions as a histone acetyltransferase (HAT). It is also an important player in acetylation of a number ...Transcriptional coactivator p300, a critical player in eukaryotic gene regulation, primarily functions as a histone acetyltransferase (HAT). It is also an important player in acetylation of a number of nonhistone proteins, p53 being the most prominent one. Recruitment of p300 to p53 is pivotal in the regulation of p53-dependent genes. Emerging evidence suggests that p300 adopts an active conformation upon binding to the tetrameric p53, resulting in its enhanced acetylation activity. As a modular protein, p300 consists of multiple well-defined domains, where the structured domains are interlinked with unstructured linker regions. A crystal structure of the central domain of p300 encompassing Bromo, RING, PHD, and HAT domains demonstrates a compact module, where the HAT active site stays occluded by the RING domain. However, although p300 has a significant role in mediating the transcriptional activity of p53, only a few structural details on the complex of these two full-length proteins are available. Here, we present a cryo-electron microscopy (cryo-EM) study on the p300-p53 complex. The three-dimensional cryo-EM density map of the p300-p53 complex, when compared to the cryo-EM map of free p300, revealed that substantial change in the relative arrangement of Bromo and HAT domains occurs upon complex formation, which is likely required for exposing HAT active site and subsequent acetyltransferase activity. Our observation correlates well with previous studies showing that the presence of Bromodomain is obligatory for effective acetyltransferase activity of HAT. Thus, our result sheds new light on the mechanism whereby p300, following binding with p53, gets activated.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJul 12, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Assembly

Deposited unit
A: Histone acetyltransferase p300
B: Cellular tumor antigen p53
C: Cellular tumor antigen p53
D: Cellular tumor antigen p53
E: Cellular tumor antigen p53


Theoretical massNumber of molelcules
Total (without water)191,6965
Polymers191,6965
Non-polymers00
Water0
1
A: Histone acetyltransferase p300


Theoretical massNumber of molelcules
Total (without water)72,1001
Polymers72,1001
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2
B: Cellular tumor antigen p53
C: Cellular tumor antigen p53
D: Cellular tumor antigen p53
E: Cellular tumor antigen p53


Theoretical massNumber of molelcules
Total (without water)119,5964
Polymers119,5964
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
DetailsOne molecule of p300 interacts with four molecules of p53 (i.e, p300 monomer interacts with p53 tetramer)

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Components

#1: Protein Histone acetyltransferase p300 / p300 HAT / E1A-associated protein p300 / Coordinate model: Cα atoms only


Mass: 72100.062 Da / Num. of mol.: 1 / Fragment: UNP residues 1046-1664
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EP300, P300 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q09472, histone acetyltransferase
#2: Protein
Cellular tumor antigen p53 / P53 / Antigen NY-CO-13 / Phosphoprotein p53 / Tumor suppressor p53 / Coordinate model: Cα atoms only


Mass: 29898.908 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TP53, P53 / Production host: Escherichia coli (E. coli) / References: UniProt: P04637

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: p300-p53 complex / Type: COMPLEX
Details: Proteins were purified separately and then complex was made for cryo-EM.
Entity ID: 1, 2 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component

Buffer-ID: 1

IDConc.NameFormula
110 mMTris-Cl
2150 mMNaclSodium chloride
35 mMMgCl2
420 microMZnCl2
52 mMPMSF
61 mMDTT
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 78894 X / Nominal defocus max: 4500 nm / Nominal defocus min: 1700 nm / Calibrated defocus min: 1700 nm / Calibrated defocus max: 4500 nm / Cs: 2 mm / C2 aperture diameter: 150 µm
Specimen holderCryogen: NITROGEN
Model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 15 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetailsFitting-ID
1EMAN22.12particle selectionparticles picked semiautomatically: using both e2boxer.py and manually.
2SPIDERparticle selectionparticles picked in EMAN2 and exported to SPIDER and also particles were picked separately in SPIDER
3TIA4.7image acquisition
5EMAN22.12CTF correction
6SPIDERCTF correction
9UCSF Chimera1.11model fittingCrystal structure 4BHW fitted using Chimera. The bromodomain matches remarkably well with the central spout. RING and PHD domains are fitted manually at the weak densities beside the bromodomain.1
12PyMOLmodel fittingp53 dimers (pdb: 3KMD) fitted manually in both the side-lobes of p53 density.2
14EMAN22.12initial Euler assignment
15SPIDERfinal Euler assignment
16SPIDERclassification
17SPIDER3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 10.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10088 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model building
ID
1
2
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-IDPdb chain residue range
14BHW

4bhw

A11046-1664
23KMD

3kmd

292-291
31C26

1c26

2325-356
RefinementHighest resolution: 10.7 Å

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