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- PDB-8sea: Cryo-EM structure of a double loaded human UBA7-UBE2L6-ISG15 thio... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8sea | ||||||
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Title | Cryo-EM structure of a double loaded human UBA7-UBE2L6-ISG15 thioester mimetic complex (Form 1) | ||||||
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![]() | LIGASE / Signaling Protein | ||||||
Function / homology | ![]() ISG15 activating enzyme activity / ISG15 transferase activity / Ligases; Forming carbon-sulfur bonds; Acid-thiol ligases / positive regulation of protein oligomerization / ISG15-protein conjugation / regulation of type II interferon production / protein localization to mitochondrion / NS1 Mediated Effects on Host Pathways / response to type I interferon / negative regulation of type I interferon-mediated signaling pathway ...ISG15 activating enzyme activity / ISG15 transferase activity / Ligases; Forming carbon-sulfur bonds; Acid-thiol ligases / positive regulation of protein oligomerization / ISG15-protein conjugation / regulation of type II interferon production / protein localization to mitochondrion / NS1 Mediated Effects on Host Pathways / response to type I interferon / negative regulation of type I interferon-mediated signaling pathway / E2 ubiquitin-conjugating enzyme / negative regulation of viral genome replication / RSV-host interactions / ubiquitin conjugating enzyme activity / positive regulation of interleukin-10 production / positive regulation of bone mineralization / ubiquitin ligase complex / negative regulation of protein ubiquitination / positive regulation of interferon-beta production / positive regulation of erythrocyte differentiation / ubiquitin binding / integrin-mediated signaling pathway / Negative regulators of DDX58/IFIH1 signaling / Termination of translesion DNA synthesis / DDX58/IFIH1-mediated induction of interferon-alpha/beta / protein modification process / response to virus / PKR-mediated signaling / ISG15 antiviral mechanism / modification-dependent protein catabolic process / protein polyubiquitination / protein tag activity / positive regulation of type II interferon production / ubiquitin-protein transferase activity / Interferon alpha/beta signaling / integrin binding / Antigen processing: Ubiquitination & Proteasome degradation / ubiquitin-dependent protein catabolic process / defense response to virus / protein ubiquitination / defense response to bacterium / Amyloid fiber formation / innate immune response / ubiquitin protein ligase binding / DNA damage response / SARS-CoV-2 activates/modulates innate and adaptive immune responses / extracellular region / nucleoplasm / ATP binding / nucleus / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | ||||||
![]() | Afsar, M. / Jia, L. / Ruben, E.A. / Olsen, S.K. | ||||||
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![]() | ![]() Title: Cryo-EM structures of Uba7 reveal the molecular basis for ISG15 activation and E1-E2 thioester transfer. Authors: Mohammad Afsar / GuanQun Liu / Lijia Jia / Eliza A Ruben / Digant Nayak / Zuberwasim Sayyad / Priscila Dos Santos Bury / Kristin E Cano / Anindita Nayak / Xiang Ru Zhao / Ankita Shukla / ...Authors: Mohammad Afsar / GuanQun Liu / Lijia Jia / Eliza A Ruben / Digant Nayak / Zuberwasim Sayyad / Priscila Dos Santos Bury / Kristin E Cano / Anindita Nayak / Xiang Ru Zhao / Ankita Shukla / Patrick Sung / Elizabeth V Wasmuth / Michaela U Gack / Shaun K Olsen / ![]() Abstract: ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin- ...ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin-like protein that is activated by an E1 enzyme (Uba7) and transferred to a cognate E2 enzyme (UBE2L6) to form a UBE2L6-ISG15 intermediate that functions with E3 ligases that catalyze conjugation of ISG15 to target proteins. Despite its biological importance, the molecular basis by which Uba7 catalyzes ISG15 activation and transfer to UBE2L6 is unknown as there is no available structure of Uba7. Here, we present cryo-EM structures of human Uba7 in complex with UBE2L6, ISG15 adenylate, and ISG15 thioester intermediate that are poised for catalysis of Uba7-UBE2L6-ISG15 thioester transfer. Our structures reveal a unique overall architecture of the complex compared to structures from the ubiquitin conjugation pathway, particularly with respect to the location of ISG15 thioester intermediate. Our structures also illuminate the molecular basis for Uba7 activities and for its exquisite specificity for ISG15 and UBE2L6. Altogether, our structural, biochemical, and human cell-based data provide significant insights into the functions of Uba7, UBE2L6, and ISG15 in cells. | ||||||
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 266.1 KB | Display | ![]() |
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PDB format | ![]() | 208.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 40408MC ![]() 8se9C ![]() 8sebC ![]() 8sv8C ![]() 40410 M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 111822.102 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() | ||||||
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#2: Protein | Mass: 17147.637 Da / Num. of mol.: 2 / Mutation: C78S Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #3: Protein | | Mass: 17643.377 Da / Num. of mol.: 1 / Mutation: C98S,C102S,L121K Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() References: UniProt: O14933, E2 ubiquitin-conjugating enzyme #4: Chemical | ChemComp-AMP / | Has ligand of interest | Y | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Cryo-EM structure of a double loaded human UBA7-UBE2L6-ISG15 thioester mimetic complex (Form 1) Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES |
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Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2888 nm / Nominal defocus min: 100 nm |
Image recording | Electron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76089 / Symmetry type: POINT | ||||||||||||||||||||||||
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