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Yorodumi- EMDB-12665: Cryo-EM structure of pre-dephosphorylation complex of phosphoryla... -
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-Basic information
Entry | Database: EMDB / ID: EMD-12665 | |||||||||
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Title | Cryo-EM structure of pre-dephosphorylation complex of phosphorylated eIF2alpha with trapped holophosphatase (PP1A_D64A/PPP1R15A/G-actin/DNase I) | |||||||||
Map data | Unsharpened GS-FSC map after corrected FSC-mask auto-tightening from non-uniform refinement in CryoSPARC. The contour level was set when opend in UCSF Chimera. | |||||||||
Sample |
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Function / homology | Function and homology information fatty acid derivative binding / positive regulation of translational initiation in response to stress / : / regulation of translational initiation by eIF2 alpha dephosphorylation / regulation of neutrophil mediated cytotoxicity / zymogen granule / regulation of acute inflammatory response / : / : / protein phosphatase type 1 complex ...fatty acid derivative binding / positive regulation of translational initiation in response to stress / : / regulation of translational initiation by eIF2 alpha dephosphorylation / regulation of neutrophil mediated cytotoxicity / zymogen granule / regulation of acute inflammatory response / : / : / protein phosphatase type 1 complex / regulation of translation in response to endoplasmic reticulum stress / translation initiation ternary complex / glial limiting end-foot / response to kainic acid / deoxyribonuclease I / HRI-mediated signaling / Cellular response to mitochondrial stress / response to manganese-induced endoplasmic reticulum stress / PTW/PP1 phosphatase complex / positive regulation of type B pancreatic cell apoptotic process / negative regulation of translational initiation in response to stress / Response of EIF2AK1 (HRI) to heme deficiency / Recycling of eIF2:GDP / PERK-mediated unfolded protein response / PERK regulates gene expression / peptidyl-serine dephosphorylation / regulation of translational initiation in response to stress / eukaryotic translation initiation factor 2 complex / : / protein localization to endoplasmic reticulum / deoxyribonuclease I activity / neutrophil activation involved in immune response / protein phosphatase regulator activity / protein phosphatase 1 binding / eukaryotic 48S preinitiation complex / DNA catabolic process / Formation of the ternary complex, and subsequently, the 43S complex / cytoskeletal motor activator activity / Ribosomal scanning and start codon recognition / myosin phosphatase activity / glycogen metabolic process / Translation initiation complex formation / protein serine/threonine phosphatase activity / tropomyosin binding / protein-serine/threonine phosphatase / myosin heavy chain binding / negative regulation of PERK-mediated unfolded protein response / mesenchyme migration / detection of maltose stimulus / troponin I binding / entrainment of circadian clock by photoperiod / maltose transport complex / protein phosphatase activator activity / filamentous actin / actin filament bundle / phosphatase activity / : / skeletal muscle thin filament assembly / actin filament bundle assembly / carbohydrate transport / striated muscle thin filament / phosphoprotein phosphatase activity / transition metal ion binding / Response of EIF2AK4 (GCN2) to amino acid deficiency / skeletal muscle myofibril / actin monomer binding / carbohydrate transmembrane transporter activity / maltose binding / mitophagy / intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress / GTP hydrolysis and joining of the 60S ribosomal subunit / maltose transport / maltodextrin transmembrane transport / L13a-mediated translational silencing of Ceruloplasmin expression / skeletal muscle fiber development / stress fiber / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / stress granule assembly / titin binding / translation initiation factor activity / actin filament polymerization / response to endoplasmic reticulum stress / protein dephosphorylation / cellular response to amino acid starvation / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / Downregulation of TGF-beta receptor signaling / filopodium / actin filament / translational initiation / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / circadian regulation of gene expression / ABC-family proteins mediated transport / PKR-mediated signaling / regulation of circadian rhythm / cytoplasmic stress granule / calcium-dependent protein binding / cellular response to UV / positive regulation of canonical Wnt signaling pathway / lamellipodium Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) / Oryctolagus cuniculus (rabbit) / Rabbit (rabbit) / Bovine (cattle) / Escherichia coli (strain K12) (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.96 Å | |||||||||
Authors | Yan Y / Hardwick S / Ron D | |||||||||
Funding support | United Kingdom, 1 items
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Citation | Journal: Nat Struct Mol Biol / Year: 2021 Title: Higher-order phosphatase-substrate contacts terminate the integrated stress response. Authors: Yahui Yan / Heather P Harding / David Ron / Abstract: Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally ...Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally affecting flexible access of the phosphopeptide to the active site. However, catalytic efficiency of holophosphatases towards their phosphoprotein substrates remains unexplained. Here we present a cryo-EM structure of the tripartite PP1c-PPP1R15A-G-actin holophosphatase that terminates signaling in the mammalian integrated stress response (ISR) in the pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin, whose essential role in eIF2α dephosphorylation is supported crystallographically, biochemically and genetically, aligns the catalytic and regulatory subunits, creating a composite surface that engages the N-terminal domain of eIF2α to position the distant phosphoserine-51 at the active site. Substrate residues that mediate affinity for the holophosphatase also make critical contacts with eIF2α kinases. Thus, a convergent process of higher-order substrate recognition specifies functionally antagonistic phosphorylation and dephosphorylation in the ISR. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_12665.map.gz | 73.8 MB | EMDB map data format | |
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Header (meta data) | emd-12665-v30.xml emd-12665.xml | 27.7 KB 27.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_12665_fsc.xml | 15.7 KB | Display | FSC data file |
Images | emd_12665.png | 205.4 KB | ||
Masks | emd_12665_msk_1.map | 149.9 MB | Mask map | |
Others | emd_12665_additional_1.map.gz emd_12665_half_map_1.map.gz emd_12665_half_map_2.map.gz | 13.4 MB 139 MB 139 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12665 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12665 | HTTPS FTP |
-Validation report
Summary document | emd_12665_validation.pdf.gz | 516.9 KB | Display | EMDB validaton report |
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Full document | emd_12665_full_validation.pdf.gz | 516.5 KB | Display | |
Data in XML | emd_12665_validation.xml.gz | 19.7 KB | Display | |
Data in CIF | emd_12665_validation.cif.gz | 25.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12665 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12665 | HTTPS FTP |
-Related structure data
Related structure data | 7nzmMC 7nxvC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_12665.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Unsharpened GS-FSC map after corrected FSC-mask auto-tightening from non-uniform refinement in CryoSPARC. The contour level was set when opend in UCSF Chimera. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.652 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_12665_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: ResolveCyroEM map; 1.81rmsd (0.443e/A^3) in COOT
File | emd_12665_additional_1.map | ||||||||||||
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Annotation | ResolveCyroEM map; 1.81rmsd (0.443e/A^3) in COOT | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half (A) map of the main map
File | emd_12665_half_map_1.map | ||||||||||||
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Annotation | half (A) map of the main map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half (B) map of the main map
File | emd_12665_half_map_2.map | ||||||||||||
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Annotation | half (B) map of the main map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : pre-dephosphorylation complex of phosphorylated eIF2alpha_2-187 w...
Entire | Name: pre-dephosphorylation complex of phosphorylated eIF2alpha_2-187 with trapped holophosphatase (PP1A_D64A/PPP1R15A_553-624/G-actin) |
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Components |
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-Supramolecule #1: pre-dephosphorylation complex of phosphorylated eIF2alpha_2-187 w...
Supramolecule | Name: pre-dephosphorylation complex of phosphorylated eIF2alpha_2-187 with trapped holophosphatase (PP1A_D64A/PPP1R15A_553-624/G-actin) type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 Details: One copy of each component was present in the complex: phosphorylated eIF2alpha_2-187, PP1A_D64A, PPP1R15A_553-624, G-actin and DNase I. The full complex was purified by size exclusion chromatography. |
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Molecular weight | Experimental: 181 KDa |
-Macromolecule #1: Eukaryotic translation initiation factor 2 subunit 1
Macromolecule | Name: Eukaryotic translation initiation factor 2 subunit 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 21.817863 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: PGLSCRFYQH KFPEVEDVVM VNVRSIAEMG AYVSLLEYNN IEGMILLSEL (SEP)RRRIRSINK LIRIGRNECV VVIRVD KEK GYIDLSKRRV SPEEAIKCED KFTKSKTVYS ILRHVAEVLE YTKDEQLESL FQRTAWVFDD KYKRPGYGAY DAFKHAV SD PSILDSLDLN EDEREVLINN INRRLTPQ |
-Macromolecule #2: Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
Macromolecule | Name: Serine/threonine-protein phosphatase PP1-alpha catalytic subunit type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: protein-serine/threonine phosphatase |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) |
Molecular weight | Theoretical: 33.647621 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: LNLDSIIGRL LEVQGSRPGK NVQLTENEIR GLCLKSREIF LSQPILLELE APLKICGAIH GQYYDLLRLF EYGGFPPESN YLFLGDYVD RGKQSLETIC LLLAYKIKYP ENFFLLRGNH ECASINRIYG FYDECKRRYN IKLWKTFTDC FNCLPIAAIV D EKIFCCHG ...String: LNLDSIIGRL LEVQGSRPGK NVQLTENEIR GLCLKSREIF LSQPILLELE APLKICGAIH GQYYDLLRLF EYGGFPPESN YLFLGDYVD RGKQSLETIC LLLAYKIKYP ENFFLLRGNH ECASINRIYG FYDECKRRYN IKLWKTFTDC FNCLPIAAIV D EKIFCCHG GLSPDLQSME QIRRIMRPTD VPDQGLLCDL LWSDPDKDVQ GWGENDRGVS FTFGAEVVAK FLHKHDLDLI CR AHQVVED GYEFFAKRQL VTLFSAPNYC GEFDNAGAMM SVDETLMCSF QILKPAD |
-Macromolecule #3: Actin, alpha skeletal muscle, intermediate form
Macromolecule | Name: Actin, alpha skeletal muscle, intermediate form / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Rabbit (rabbit) |
Molecular weight | Theoretical: 41.862613 KDa |
Sequence | String: DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIITNWD DMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSGDGV T HNVPIYEG ...String: DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIITNWD DMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSGDGV T HNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSLEK SY ELPDGQV ITIGNERFRC PETLFQPSFI GMESAGIHET TYNSIMKCDI DIRKDLYANN VMSGGTTMYP GIADRMQKEI TAL APSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ITKQEYDEAG PSIVHRKCF |
-Macromolecule #4: Deoxyribonuclease-1
Macromolecule | Name: Deoxyribonuclease-1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: deoxyribonuclease I |
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Source (natural) | Organism: Bovine (cattle) |
Molecular weight | Theoretical: 29.092574 KDa |
Sequence | String: LKIAAFNIRT FGETKMSNAT LASYIVRIVR RYDIVLIQEV RDSHLVAVGK LLDYLNQDDP NTYHYVVSEP LGRNSYKERY LFLFRPNKV SVLDTYQYDD GCESCGNDSF SREPAVVKFS SHSTKVKEFA IVALHSAPSD AVAEINSLYD VYLDVQQKWH L NDVMLMGD ...String: LKIAAFNIRT FGETKMSNAT LASYIVRIVR RYDIVLIQEV RDSHLVAVGK LLDYLNQDDP NTYHYVVSEP LGRNSYKERY LFLFRPNKV SVLDTYQYDD GCESCGNDSF SREPAVVKFS SHSTKVKEFA IVALHSAPSD AVAEINSLYD VYLDVQQKWH L NDVMLMGD FNADCSYVTS SQWSSIRLRT SSTFQWLIPD SADTTATSTN CAYDRIVVAG SLLQSSVVPG SAAPFDFQAA YG LSNEMAL AISDHYPVEV TLT |
-Macromolecule #5: Protein phosphatase 1 regulatory subunit 15A,Maltose/maltodextrin...
Macromolecule | Name: Protein phosphatase 1 regulatory subunit 15A,Maltose/maltodextrin-binding periplasmic protein type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12 |
Molecular weight | Theoretical: 49.169797 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: ARKVRFSEKV TVHFLAVWAG PAQAARQGPW EQLARDRSRF ARRITQAQEE LSPCLTPAAR ARAWARLRNP PLQAKIEEGK LVIWINGDK GYNGLAEVGK KFEKDTGIKV TVEHPDKLEE KFPQVAATGD GPDIIFWAHD RFGGYAQSGL LAEITPDKAF Q DKLYPFTW ...String: ARKVRFSEKV TVHFLAVWAG PAQAARQGPW EQLARDRSRF ARRITQAQEE LSPCLTPAAR ARAWARLRNP PLQAKIEEGK LVIWINGDK GYNGLAEVGK KFEKDTGIKV TVEHPDKLEE KFPQVAATGD GPDIIFWAHD RFGGYAQSGL LAEITPDKAF Q DKLYPFTW DAVRYNGKLI AYPIAVEALS LIYNKDLLPN PPKTWEEIPA LDKELKAKGK SALMFNLQEP YFTWPLIAAD GG YAFKYEN GKYDIKDVGV DNAGAKAGLT FLVDLIKNKH MNADTDYSIA EAAFNKGETA MTINGPWAWS NIDTSKVNYG VTV LPTFKG QPSKPFVGVL SAGINAASPN KELAKEFLEN YLLTDEGLEA VNKDKPLGAV ALKSYEEELA KDPRIAATME NAQK GEIMP NIPQMSAFWY AVRTAVINAA SGRQTVDEAL KDAQTRITK |
-Macromolecule #7: MANGANESE (II) ION
Macromolecule | Name: MANGANESE (II) ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MN |
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Molecular weight | Theoretical: 54.938 Da |
-Macromolecule #8: ADENOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 1 / Formula: ATP |
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Molecular weight | Theoretical: 507.181 Da |
Chemical component information | ChemComp-ATP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 5 mg/mL | ||||||||||||||||
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Buffer | pH: 7.4 Component:
Details: 0.22mM Triton X-100 was added into the solution before plunging. | ||||||||||||||||
Grid | Model: UltrAuFoil R0.6/1 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER / Details: current 25mA at Pelco EasiGLOW | ||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Phase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 4025 / Average electron dose: 46.84 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -2.8000000000000003 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: OTHER / Overall B value: 47 | ||||||||||
Output model | PDB-7nzm: |