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- EMDB-44954: Human Vault Cage in complex with NAD+ -

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Basic information

Entry
Database: EMDB / ID: EMD-44954
TitleHuman Vault Cage in complex with NAD+
Map dataSharpened cryo-EM map of the human MVP cage with bound NAD
Sample
  • Complex: Binary complex of NAD+ bound to oligomerized human MVP
    • Protein or peptide: Major Vault Protein
KeywordsVault / Vault Cage / MVP / Major Vault Protein / SPFH / PARP4 / Poly(ADP-ribose)Polymerase 4 / MINT / NAD+ / NAD / Poly(ADP-ribose)Polymerase / Ribonucleoprotein / Megadalton complex / TEP1 / vault RNA / PROTEIN TRANSPORT
Function / homology
Function and homology information


negative regulation of protein tyrosine kinase activity / protein activation cascade / negative regulation of protein autophosphorylation / ERBB signaling pathway / negative regulation of epidermal growth factor receptor signaling pathway / mRNA transport / nuclear pore / protein transport / protein phosphatase binding / secretory granule lumen ...negative regulation of protein tyrosine kinase activity / protein activation cascade / negative regulation of protein autophosphorylation / ERBB signaling pathway / negative regulation of epidermal growth factor receptor signaling pathway / mRNA transport / nuclear pore / protein transport / protein phosphatase binding / secretory granule lumen / ficolin-1-rich granule lumen / cytoskeleton / cell population proliferation / ribonucleoprotein complex / Neutrophil degranulation / protein kinase binding / perinuclear region of cytoplasm / extracellular exosome / extracellular region / identical protein binding / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
Major vault protein, N-terminal / Major vault protein, shoulder domain / Major vault protein / Major vault protein repeat domain 3 / Major vault protein repeat domain 2 / Major vault protein repeat domain 4 / Major vault protein repeat domain / Major vault protein repeat domain superfamily / Major vault protein repeat domain 2 superfamily / Major Vault Protein repeat domain ...Major vault protein, N-terminal / Major vault protein, shoulder domain / Major vault protein / Major vault protein repeat domain 3 / Major vault protein repeat domain 2 / Major vault protein repeat domain 4 / Major vault protein repeat domain / Major vault protein repeat domain superfamily / Major vault protein repeat domain 2 superfamily / Major Vault Protein repeat domain / Shoulder domain / Major Vault Protein repeat domain / Major Vault Protein Repeat domain / Major Vault Protein repeat domain / MVP (vault) repeat profile. / Band 7/SPFH domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLodwick JE / Zhao M
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM143052 United States
CitationJournal: bioRxiv / Year: 2024
Title: Structural Insights into the Roles of PARP4 and NAD in the Human Vault Cage.
Abstract: Vault is a massive ribonucleoprotein complex found across Eukaryota. The major vault protein (MVP) oligomerizes into an ovular cage, which contains several minor vault components (MVCs) and is ...Vault is a massive ribonucleoprotein complex found across Eukaryota. The major vault protein (MVP) oligomerizes into an ovular cage, which contains several minor vault components (MVCs) and is thought to transport transiently bound "cargo" molecules. Vertebrate vaults house a poly (ADP-ribose) polymerase (known as PARP4 in humans), which is the only MVC with known enzymatic activity. Despite being discovered decades ago, the molecular basis for PARP4's interaction with MVP remains unclear. In this study, we determined the structure of the human vault cage in complex with PARP4 and its enzymatic substrate NAD . The structures reveal atomic-level details of the protein-binding interface, as well as unexpected NAD -binding pockets within the interior of the vault cage. In addition, proteomics data show that human vaults purified from wild-type and PARP4-depleted cells interact with distinct subsets of proteins. Our results thereby support a model in which PARP4's specific incorporation into the vault cage helps to regulate vault's selection of cargo and its subcellular localization. Further, PARP4's proximity to MVP's NAD -binding sites could support its enzymatic function within the vault.
History
DepositionMay 20, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateFeb 5, 2025-
Current statusFeb 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44954.png

Downloads & links

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Map

FileDownload / File: emd_44954.map.gz / Format: CCP4 / Size: 1.9 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM map of the human MVP cage with bound NAD
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 800 pix.
= 1075.2 Å		1.34 Å/pix.
x 800 pix.
= 1075.2 Å		1.34 Å/pix.
x 800 pix.
= 1075.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.344 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.9912067 - 2.2005427
Average (Standard dev.)-0.00020358516 (±0.049165573)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions800800800
Spacing800800800
CellA=B=C: 1075.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened cryo-EM map of the human MVP cage with bound NAD

Fileemd_44954_additional_1.map
AnnotationUnsharpened cryo-EM map of the human MVP cage with bound NAD
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM half map of the human MVP cage with bound NAD

Fileemd_44954_half_map_1.map
AnnotationCryo-EM half map of the human MVP cage with bound NAD
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM half map of the human MVP cage with bound NAD

Fileemd_44954_half_map_2.map
AnnotationCryo-EM half map of the human MVP cage with bound NAD
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Binary complex of NAD+ bound to oligomerized human MVP

EntireName: Binary complex of NAD+ bound to oligomerized human MVP
Components
  • Complex: Binary complex of NAD+ bound to oligomerized human MVP
    • Protein or peptide: Major Vault Protein

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Supramolecule #1: Binary complex of NAD+ bound to oligomerized human MVP

SupramoleculeName: Binary complex of NAD+ bound to oligomerized human MVP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.8 MDa

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Macromolecule #1: Major Vault Protein

MacromoleculeName: Major Vault Protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MATEEFIIRI PPYHYIHVLD QNSNVSRVEV GPKTYIRQDN ERVLFAPMRM VTVPPRHYCT VANPVSRDAQ GLVLFDVTGQ VRLRHADLE IRLAQDPFPL YPGEVLEKDI TPLQVVLPNT ALHLKALLDF EDKDGDKVVA GDEWLFEGPG TYIPRKEVEV V EIIQATII ...String:
MATEEFIIRI PPYHYIHVLD QNSNVSRVEV GPKTYIRQDN ERVLFAPMRM VTVPPRHYCT VANPVSRDAQ GLVLFDVTGQ VRLRHADLE IRLAQDPFPL YPGEVLEKDI TPLQVVLPNT ALHLKALLDF EDKDGDKVVA GDEWLFEGPG TYIPRKEVEV V EIIQATII RQNQALRLRA RKECWDRDGK ERVTGEEWLV TTVGAYLPAV FEEVLDLVDA VILTEKTALH LRARRNFRDF RG VSRRTGE EWLVTVQDTE AHVPDVHEEV LGVVPITTLG PHNYCVILDP VGPDGKNQLG QKRVVKGEKS FFLQPGEQLE QGI QDVYVL SEQQGLLLRA LQPLEEGEDE EKVSHQAGDH WLIRGPLEYV PSAKVEVVEE RQAIPLDENE GIYVQDVKTG KVRA VIGST YMLTQDEVLW EKELPPGVEE LLNKGQDPLA DRGEKDTAKS LQPLAPRNKT RVVSYRVPHN AAVQVYDYRE KRARV VFGP ELVSLGPEEQ FTVLSLSAGR PKRPHARRAL CLLLGPDFFT DVITIETADH ARLQLQLAYN WHFEVNDRKD PQETAK LFS VPDFVGDACK AIASRVRGAV ASVTFDDFHK NSARIIRTAV FGFETSEAKG PDGMALPRPR DQAVFPQNGL VVSSVDV QS VEPVDQRTRD ALQRSVQLAI EITTNSQEAA AKHEAQRLEQ EARGRLERQK ILDQSEAEKA RKELLELEAL SMAVESTG T AKAEAESRAE AARIEGEGSV LQAKLKAQAL AIETEAELQR VQKVRELELV YARAQLELEV SKAQQLAEVE VKKFKQMTE AIGPSTIRDL AVAGPEMQVK LLQSLGLKST LITDGSTPIN LFNTAFGLLG MGPEGQPLGR RVASGPSPGE GISPQSAQAP QAPGDNHVV PVLR

UniProtKB: Major vault protein

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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

Concentration1.8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
5.0 mMMgCl2Magnesium chloride
5.0 mMCaCl2Calcium chloride
0.25 mMC4H10O2S2DTT

Details: 50 mM HEPES, 5 mM MgCl2, 5 mM CaCl2, 0.25 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 14 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
Details: Quantifoil grids with a 2 nm continuous carbon coating were subjected to a 15 s, 5W plasma cleaning program in O2 gas
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Details: Vitrification carried out under standard conditions.
DetailsSample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 4952 / Average electron dose: 45.0 e/Å2
Details: Images were collected in movie mode, with 40 frames per image.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 318989
Startup modelType of model: OTHER / Details: EM map from a previous dataset we collected
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D39 (2x39 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1)
Software - details: cryoSPARC Non-Uniform Refinement was performed. The program was instructed to optimize the per-particle defocus and per-exposure-group CTF parameters, fit the anisotropic beam ...Software - details: cryoSPARC Non-Uniform Refinement was performed. The program was instructed to optimize the per-particle defocus and per-exposure-group CTF parameters, fit the anisotropic beam magnification, and apply Ewald sphere correction using the correct curvature sign.
Number images used: 108991
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationNumber classes: 50 / Avg.num./class: 2180 / Software - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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