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Yorodumi- EMDB-2981: Cryo-EM reveals the conformation of a substrate analogue in the h... -
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-Basic information
Entry | Database: EMDB / ID: EMD-2981 | |||||||||
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Title | Cryo-EM reveals the conformation of a substrate analogue in the human 20S proteasome core | |||||||||
Map data | reconstruction of the human 20S proteasome core, as determined by the 3D reconstitution algorithm, without further masking, sharpening or Fourier filtering | |||||||||
Sample |
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Keywords | proteasome / 20S / human / AdaAhx3L3VS / ligand / inhibitor / drug design | |||||||||
Function / homology | Function and homology information purine ribonucleoside triphosphate binding / regulation of endopeptidase activity / Regulation of ornithine decarboxylase (ODC) / proteasome core complex / Cross-presentation of soluble exogenous antigens (endosomes) / Somitogenesis / immune system process / myofibril / NF-kappaB binding / proteasome endopeptidase complex ...purine ribonucleoside triphosphate binding / regulation of endopeptidase activity / Regulation of ornithine decarboxylase (ODC) / proteasome core complex / Cross-presentation of soluble exogenous antigens (endosomes) / Somitogenesis / immune system process / myofibril / NF-kappaB binding / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / : / negative regulation of inflammatory response to antigenic stimulus / response to organonitrogen compound / sarcomere / proteolysis involved in protein catabolic process / proteasome complex / Regulation of activated PAK-2p34 by proteasome mediated degradation / ciliary basal body / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / Asymmetric localization of PCP proteins / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / Ubiquitin-dependent degradation of Cyclin D / AUF1 (hnRNP D0) binds and destabilizes mRNA / Degradation of DVL / TNFR2 non-canonical NF-kB pathway / Assembly of the pre-replicative complex / Vpu mediated degradation of CD4 / proteasomal protein catabolic process / P-body / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Dectin-1 mediated noncanonical NF-kB signaling / Hh mutants are degraded by ERAD / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Degradation of GLI1 by the proteasome / lipopolysaccharide binding / Degradation of AXIN / Defective CFTR causes cystic fibrosis / Activation of NF-kappaB in B cells / Negative regulation of NOTCH4 signaling / Hedgehog ligand biogenesis / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Vif-mediated degradation of APOBEC3G / G2/M Checkpoints / Hedgehog 'on' state / Autodegradation of the E3 ubiquitin ligase COP1 / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / Regulation of RUNX3 expression and activity / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / response to virus / MAPK6/MAPK4 signaling / Degradation of beta-catenin by the destruction complex / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / ABC-family proteins mediated transport / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / response to organic cyclic compound / CDK-mediated phosphorylation and removal of Cdc6 / CLEC7A (Dectin-1) signaling / SCF(Skp2)-mediated degradation of p27/p21 / Regulation of expression of SLITs and ROBOs / nuclear matrix / FCERI mediated NF-kB activation / Interleukin-1 signaling / Regulation of PTEN stability and activity / Orc1 removal from chromatin / Regulation of RAS by GAPs / Separation of Sister Chromatids / Regulation of RUNX2 expression and activity / UCH proteinases / The role of GTSE1 in G2/M progression after G2 checkpoint / KEAP1-NFE2L2 pathway / Antigen processing: Ubiquitination & Proteasome degradation / Downstream TCR signaling / Neddylation / RUNX1 regulates transcription of genes involved in differentiation of HSCs / positive regulation of NF-kappaB transcription factor activity / peptidase activity / ER-Phagosome pathway / regulation of inflammatory response / postsynapse / proteasome-mediated ubiquitin-dependent protein catabolic process / response to oxidative stress / secretory granule lumen / endopeptidase activity / ficolin-1-rich granule lumen / nuclear body / Ub-specific processing proteases / cadherin binding / intracellular membrane-bounded organelle / centrosome / synapse / ubiquitin protein ligase binding / Neutrophil degranulation / mitochondrion / proteolysis Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
Authors | da Fonseca PCA / Morris EP | |||||||||
Citation | Journal: Nat Commun / Year: 2015 Title: Cryo-EM reveals the conformation of a substrate analogue in the human 20S proteasome core. Authors: Paula C A da Fonseca / Edward P Morris / Abstract: The proteasome is a highly regulated protease complex fundamental for cell homeostasis and controlled cell cycle progression. It functions by removing a wide range of specifically tagged proteins, ...The proteasome is a highly regulated protease complex fundamental for cell homeostasis and controlled cell cycle progression. It functions by removing a wide range of specifically tagged proteins, including key cellular regulators. Here we present the structure of the human 20S proteasome core bound to a substrate analogue inhibitor molecule, determined by electron cryo-microscopy (cryo-EM) and single-particle analysis at a resolution of around 3.5 Å. Our map allows the building of protein coordinates as well as defining the location and conformation of the inhibitor at the different active sites. These results open new prospects to tackle the proteasome functional mechanisms. Moreover, they also further demonstrate that cryo-EM is emerging as a realistic approach for general structural studies of protein-ligand interactions. | |||||||||
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_2981.map.gz | 58.3 MB | EMDB map data format | |
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Header (meta data) | emd-2981-v30.xml emd-2981.xml | 24.6 KB 24.6 KB | Display Display | EMDB header |
Images | emd_2981.png | 152.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2981 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2981 | HTTPS FTP |
-Related structure data
Related structure data | 5a0qMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10038 (Title: Cryo-EM reveals the conformation of a substrate analogue in the human 20S proteasome core Data size: 579.1 Data #1: raw micrographs of the human 20S proteasome core complex bound to the ligand AdaAhx3L3VS [micrographs - multiframe]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2981.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | reconstruction of the human 20S proteasome core, as determined by the 3D reconstitution algorithm, without further masking, sharpening or Fourier filtering | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.04 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
+Entire : human 20S proteasome core
+Supramolecule #1000: human 20S proteasome core
+Macromolecule #1: Proteasome subunit alpha type-6
+Macromolecule #2: Proteasome subunit alpha type-2
+Macromolecule #3: Proteasome subunit alpha type-4
+Macromolecule #4: Proteasome subunit alpha type-7
+Macromolecule #5: Proteasome subunit alpha type-5
+Macromolecule #6: Proteasome subunit alpha type-1
+Macromolecule #7: Proteasome subunit alpha type-3
+Macromolecule #8: Proteasome subunit beta type-6
+Macromolecule #9: Proteasome subunit beta type-7
+Macromolecule #10: Proteasome subunit beta type-3
+Macromolecule #11: Proteasome subunit beta type-2
+Macromolecule #12: Proteasome subunit beta type-5
+Macromolecule #13: Proteasome subunit beta type-1
+Macromolecule #14: Proteasome subunit beta type-4
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 7.5 / Details: 50 mM Tris-HCl, 5 mM MgCl2 and 1mM dithiotreitol |
Grid | Details: 1.2/1.3 Quantifoil coated with freshly floated thin layer of carbon, glow discharged in amylamine atmosphere |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK III / Method: blot 2.5 seconds before plunging |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 134461 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 75000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Temperature | Average: 85 K |
Alignment procedure | Legacy - Astigmatism: objective lens astigmatism was corrected at the recording magnification |
Details | Each exposure was recorded as 17 individual frames captured at a rate of 0.056 second/frame, with an electron dose of 2.8 electrons/square angstrom. Data-set recorded using EPU software. |
Date | Feb 4, 2014 |
Image recording | Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 960 / Average electron dose: 48 e/Å2 Details: each image is the sum of 17 frames recorded by the direct electron detector |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: full recorded image |
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Final angle assignment | Details: Beta 0 degrees, gamma 90 degrees (IMAGIC) |
Final reconstruction | Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: OTHER / Software - Name: Spider, Tigris, Imagic Details: the analysis was done using a data-set recorded during a single EM session Number images used: 76500 |
Details | automatic particle picking followed by careful manual removal of false positives and addition of false negatives; high resolution analysis was done using the sum of frames 3-10 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H / Chain - #8 - Chain ID: I / Chain - #9 - Chain ID: J / Chain - #10 - Chain ID: K / Chain - #11 - Chain ID: L / Chain - #12 - Chain ID: M / Chain - #13 - Chain ID: N / Chain - #14 - Chain ID: O / Chain - #15 - Chain ID: P / Chain - #16 - Chain ID: Q / Chain - #17 - Chain ID: R / Chain - #18 - Chain ID: S / Chain - #19 - Chain ID: T / Chain - #20 - Chain ID: U / Chain - #21 - Chain ID: V / Chain - #22 - Chain ID: W / Chain - #23 - Chain ID: X / Chain - #24 - Chain ID: Y / Chain - #25 - Chain ID: Z / Chain - #26 - Chain ID: a / Chain - #27 - Chain ID: b |
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Software | Name: Coot, Phenix |
Details | The model of the human 20S proteasome core was built based on the X-ray crystal structure of the mouse constitutive apo 20S proteasome core (3UNE) using real space refinement in Coot and Phenix |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-5a0q: |