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- EMDB-30634: Cryo-EM structure of Ornithine transcarbamylase fused with Ubiqui... -

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Basic information

Entry
Database: EMDB / ID: EMD-30634
TitleCryo-EM structure of Ornithine transcarbamylase fused with Ubiquitin in complex with Ubiquitin-carboxy-hydrolase-L1 crosslinked with BS3
Map data
Sample
  • Complex: A scaffold protein of OTC-Ub in complex with 2 UCHL1s
    • Complex: OTC-Ub scaffold
    • Complex: UCHL1
Function / homology
Function and homology information


axon target recognition / male germ cell proliferation / ornithine carbamoyltransferase / alpha-2A adrenergic receptor binding / ornithine carbamoyltransferase activity / citrulline biosynthetic process / arginine biosynthetic process via ornithine / muscle cell development / neuron projection terminus / adult walking behavior ...axon target recognition / male germ cell proliferation / ornithine carbamoyltransferase / alpha-2A adrenergic receptor binding / ornithine carbamoyltransferase activity / citrulline biosynthetic process / arginine biosynthetic process via ornithine / muscle cell development / neuron projection terminus / adult walking behavior / neuromuscular process / amino acid binding / eating behavior / axonal transport of mitochondrion / protein deubiquitination / regulation of macroautophagy / axon cytoplasm / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Constitutive Signaling by NOTCH1 HD Domain Mutants / NOTCH2 Activation and Transmission of Signal to the Nucleus / Endosomal Sorting Complex Required For Transport (ESCRT) / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / Regulation of FZD by ubiquitination / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Negative regulation of FLT3 / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Downregulation of ERBB4 signaling / p75NTR recruits signalling complexes / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / APC-Cdc20 mediated degradation of Nek2A / PINK1-PRKN Mediated Mitophagy / TRAF6-mediated induction of TAK1 complex within TLR4 complex / InlA-mediated entry of Listeria monocytogenes into host cells / Pexophagy / Regulation of innate immune responses to cytosolic DNA / VLDLR internalisation and degradation / Downregulation of ERBB2:ERBB3 signaling / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / Translesion synthesis by REV1 / NF-kB is activated and signals survival / Regulation of PTEN localization / Translesion synthesis by POLK / Regulation of BACH1 activity / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Downregulation of TGF-beta receptor signaling / Josephin domain DUBs / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / negative regulation of MAP kinase activity / Regulation of activated PAK-2p34 by proteasome mediated degradation / InlB-mediated entry of Listeria monocytogenes into host cell / IKK complex recruitment mediated by RIP1 / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Autodegradation of Cdh1 by Cdh1:APC/C / TNFR1-induced NF-kappa-B signaling pathway / ubiquitin binding / APC/C:Cdc20 mediated degradation of Securin / Asymmetric localization of PCP proteins / TCF dependent signaling in response to WNT / SCF-beta-TrCP mediated degradation of Emi1 / Regulation of NF-kappa B signaling / NIK-->noncanonical NF-kB signaling / Ubiquitin-dependent degradation of Cyclin D / AUF1 (hnRNP D0) binds and destabilizes mRNA / Negative regulators of DDX58/IFIH1 signaling / NOTCH3 Activation and Transmission of Signal to the Nucleus / TNFR2 non-canonical NF-kB pathway / activated TAK1 mediates p38 MAPK activation / Assembly of the pre-replicative complex / response to ischemia / Vpu mediated degradation of CD4 / Deactivation of the beta-catenin transactivating complex / Degradation of DVL / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Recognition of DNA damage by PCNA-containing replication complex / Regulation of signaling by CBL / Dectin-1 mediated noncanonical NF-kB signaling / Hh mutants are degraded by ERAD / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Fanconi Anemia Pathway / Negative regulation of FGFR3 signaling
Similarity search - Function
Ubiquitin carboxyl-terminal hydrolase family 1 cysteine active-site. / Ornithine carbamoyltransferase / Peptidase C12, ubiquitin carboxyl-terminal hydrolase superfamily / Ubiquitin carboxyl-terminal hydrolase, family 1 / Peptidase C12, ubiquitin carboxyl-terminal hydrolase / Ornithine/putrescine carbamoyltransferase / Aspartate and ornithine carbamoyltransferases signature. / Aspartate/ornithine carbamoyltransferase / Aspartate/ornithine carbamoyltransferase, Asp/Orn-binding domain / Aspartate/ornithine carbamoyltransferase, carbamoyl-P binding ...Ubiquitin carboxyl-terminal hydrolase family 1 cysteine active-site. / Ornithine carbamoyltransferase / Peptidase C12, ubiquitin carboxyl-terminal hydrolase superfamily / Ubiquitin carboxyl-terminal hydrolase, family 1 / Peptidase C12, ubiquitin carboxyl-terminal hydrolase / Ornithine/putrescine carbamoyltransferase / Aspartate and ornithine carbamoyltransferases signature. / Aspartate/ornithine carbamoyltransferase / Aspartate/ornithine carbamoyltransferase, Asp/Orn-binding domain / Aspartate/ornithine carbamoyltransferase, carbamoyl-P binding / Aspartate/ornithine carbamoyltransferase superfamily / Aspartate/ornithine carbamoyltransferase, Asp/Orn binding domain / Aspartate/ornithine carbamoyltransferase, carbamoyl-P binding domain / Papain-like cysteine peptidase superfamily / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin domain signature. / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Ornithine carbamoyltransferase subunit I / Ubiquitin carboxyl-terminal hydrolase isozyme L1 / Polyubiquitin-C
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsChiu YH / Draczkowski P / Hsu STD
Funding support Taiwan, 3 items
OrganizationGrant numberCountry
Academia Sinica (Taiwan)AS-CFII108-111 Taiwan
Academia Sinica (Taiwan)AS-CFII-108-110 Taiwan
Academia Sinica (Taiwan)AS-CDA-109-L08 Taiwan
CitationJournal: Biochemistry / Year: 2021
Title: Direct Visualization of a 26 kDa Protein by Cryo-Electron Microscopy Aided by a Small Scaffold Protein.
Authors: Yi-Hsiang Chiu / Kuang-Ting Ko / Tzu-Jing Yang / Kuen-Phon Wu / Meng-Ru Ho / Piotr Draczkowski / Shang-Te Danny Hsu /
Abstract: Cryo-electron microscopy (cryo-EM)-based structure determination of small proteins is hindered by the technical challenges associated with low signal-to-noise ratios of their particle images in ...Cryo-electron microscopy (cryo-EM)-based structure determination of small proteins is hindered by the technical challenges associated with low signal-to-noise ratios of their particle images in intrinsically noisy micrographs. One solution is to attach the target protein to a large protein scaffold to increase its apparent size and, therefore, image contrast. Here we report a novel scaffold design based on a trimeric helical protein, ornithine transcarbamylase (OTC), fused to human ubiquitin. As a proof of principle, we demonstrated the ability to resolve a cryo-EM map of a 26 kDa human ubiquitin C-terminal hydrolase (UCHL1) attached to the C-terminus of ubiquitin as part of the trimeric assembly. The results revealed conformational changes in UCHL1 upon binding to ubiquitin, namely, a significant displacement of α-helix 2, which was also observed by X-ray crystallography. Our findings demonstrated the potential of the trimeric OTC scaffold design for studying a large number of ubiquitin interacting proteins by cryo-EM.
History
DepositionOct 23, 2020-
Header (metadata) releaseMay 19, 2021-
Map releaseMay 19, 2021-
UpdateMay 19, 2021-
Current statusMay 19, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30634.png

Downloads & links

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Map

FileDownload / File: emd_30634.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.009 / Movie #1: 0.009
Minimum - Maximum-0.008382858 - 0.024231365
Average (Standard dev.)2.1748483e-06 (±0.00084095565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z328.000328.000328.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ512512512
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0080.0240.000

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Supplemental data

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Sample components

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Entire : A scaffold protein of OTC-Ub in complex with 2 UCHL1s

EntireName: A scaffold protein of OTC-Ub in complex with 2 UCHL1s
Components
  • Complex: A scaffold protein of OTC-Ub in complex with 2 UCHL1s
    • Complex: OTC-Ub scaffold
    • Complex: UCHL1

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Supramolecule #1: A scaffold protein of OTC-Ub in complex with 2 UCHL1s

SupramoleculeName: A scaffold protein of OTC-Ub in complex with 2 UCHL1s / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3)
Molecular weightExperimental: 24.8 KDa

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Supramolecule #2: OTC-Ub scaffold

SupramoleculeName: OTC-Ub scaffold / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3)

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Supramolecule #3: UCHL1

SupramoleculeName: UCHL1 / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3)

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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

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2Tris(hydroxymethyl)aminomethane
125.0 mMNaClSodium chlorideSodium chloride
0.5 mMC9H15O6PTris(2carboxyethyl)phosphine
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was cross-linked with BS3 and treated with 0.005% of Tween20.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.0 µm / Calibrated defocus min: 0.9 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.2 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 0.97 sec. / Average electron dose: 58.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 695542
CTF correctionSoftware - Name: RELION (ver. 3.0)
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.0)
Final 3D classificationNumber classes: 5 / Avg.num./class: 42000 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 53261

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