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- EMDB-40799: Cryo-EM consensus map of a double loaded human UBA7-UBE2L6-ISG15 ... -

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Entry
Database: EMDB / ID: EMD-40799
TitleCryo-EM consensus map of a double loaded human UBA7-UBE2L6-ISG15 thioester mimetic complex
Map data
Sample
  • Complex: Double loaded human UBA7-UBE2L6-ISG15 thioester mimetic complex
Keywordsubiquitin / SIGNALING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.67 Å
AuthorsAfsar M / Jia L / Ruben EA / Olsen SK
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Commun / Year: 2023
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.
History
DepositionMay 17, 2023-
Header (metadata) releaseMar 27, 2024-
Map releaseMar 27, 2024-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40799.png

Downloads & links

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Map

FileDownload / File: emd_40799.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8332 Å
Density
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.20063601 - 0.50981253
Average (Standard dev.)-0.000101506776 (±0.011675183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 299.952 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40799_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

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Half map: #1

Fileemd_40799_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_40799_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Double loaded human UBA7-UBE2L6-ISG15 thioester mimetic complex

EntireName: Double loaded human UBA7-UBE2L6-ISG15 thioester mimetic complex
Components
  • Complex: Double loaded human UBA7-UBE2L6-ISG15 thioester mimetic complex

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Supramolecule #1: Double loaded human UBA7-UBE2L6-ISG15 thioester mimetic complex

SupramoleculeName: Double loaded human UBA7-UBE2L6-ISG15 thioester mimetic complex
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.888 µm / Nominal defocus min: 0.1 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 225140

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