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- PDB-5fki: Pseudorabies virus (PrV) nuclear egress complex proteins fitted a... -

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Basic information

Entry
Database: PDB / ID: 5fki
TitlePseudorabies virus (PrV) nuclear egress complex proteins fitted as a hexameric lattice into a sub-tomogram average derived from focused- ion beam milled lamellae electron cryo-microscopic data
Components
  • UL31
  • UL34 protein
KeywordsVIRAL PROTEIN / Alpha-Herpesvirinae / herpesvirus simplex / HSV-1 / Pseudorabies virus / PRV / Nuclear egress complex / nuclear envelope / nucleoplasmic reticulum / inner nuclear membrane / vesicle transport / nucleo-cytoplasmic transport / RYOFIB / Focused ion beam milling / FIB-SEM
Function / homology
Function and homology information


host cell nuclear inner membrane / viral budding from nuclear membrane / membrane / metal ion binding
Similarity search - Function
Herpesvirus viron egress-type / Herpesvirus virion protein U34 / Herpesvirus UL31 / Herpesvirus UL31-like protein
Similarity search - Domain/homology
UL34 protein / Nuclear egress lamina protein
Similarity search - Component
Biological speciesSuid herpesvirus 1
MethodELECTRON MICROSCOPY / electron tomography / Resolution: 35 Å
AuthorsHagen, C. / Dent, K.C. / Zeev Ben Mordehai, T. / Vasishtan, D. / Antonin, W. / Mettenleiter, T.C. / Gruenewald, K.
CitationJournal: Cell Rep / Year: 2015
Title: Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling.
Authors: Tzviya Zeev-Ben-Mordehai / Marion Weberruß / Michael Lorenz / Juliana Cheleski / Teresa Hellberg / Cathy Whittle / Kamel El Omari / Daven Vasishtan / Kyle C Dent / Karl Harlos / Kati ...Authors: Tzviya Zeev-Ben-Mordehai / Marion Weberruß / Michael Lorenz / Juliana Cheleski / Teresa Hellberg / Cathy Whittle / Kamel El Omari / Daven Vasishtan / Kyle C Dent / Karl Harlos / Kati Franzke / Christoph Hagen / Barbara G Klupp / Wolfram Antonin / Thomas C Mettenleiter / Kay Grünewald /
Abstract: Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus ...Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC), which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling.
History
DepositionOct 16, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 16, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 1.2Jun 20, 2018Group: Advisory / Data collection / Category: pdbx_unobs_or_zero_occ_atoms
Revision 1.3Nov 21, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_conn
Revision 1.4Jul 3, 2019Group: Advisory / Data collection ...Advisory / Data collection / Derived calculations / Source and taxonomy
Category: database_PDB_rev / database_PDB_rev_record ...database_PDB_rev / database_PDB_rev_record / entity_src_gen / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_conn
Item: _entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name ..._entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name / _entity_src_gen.pdbx_host_org_strain / _entity_src_gen.pdbx_host_org_variant
Revision 1.5Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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  • Simplified surface model + fitted atomic model
  • EMDB-3197
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  • EMDB-3197
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Structure viewerMolecule:
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Assembly

Deposited unit
1A: UL31
1B: UL34 protein
1C: UL31
1D: UL34 protein
1E: UL31
1F: UL34 protein
1G: UL31
1H: UL34 protein
1I: UL31
1J: UL34 protein
1K: UL31
1L: UL34 protein
1M: UL31
1N: UL34 protein
1O: UL31
1P: UL34 protein
1Q: UL31
1R: UL34 protein
1S: UL31
1T: UL34 protein
1U: UL31
1V: UL34 protein
1W: UL31
1X: UL34 protein
1Y: UL31
1Z: UL34 protein
10: UL31
11: UL34 protein
12: UL31
13: UL34 protein
14: UL31
15: UL34 protein
16: UL31
17: UL34 protein
18: UL31
19: UL34 protein
2A: UL31
2B: UL34 protein
2C: UL31
2D: UL34 protein
2E: UL31
2F: UL34 protein
2G: UL31
2H: UL34 protein
2I: UL31
2J: UL34 protein
2K: UL31
2L: UL34 protein
2M: UL31
2N: UL34 protein
2O: UL31
2P: UL34 protein
2Q: UL31
2R: UL34 protein
2S: UL31
2T: UL34 protein
2U: UL31
2V: UL34 protein
2W: UL31
2X: UL34 protein
2Y: UL31
2Z: UL34 protein
20: UL31
21: UL34 protein
22: UL31
23: UL34 protein
24: UL31
25: UL34 protein
26: UL31
27: UL34 protein
28: UL31
29: UL34 protein
3A: UL31
3B: UL34 protein
3C: UL31
3D: UL34 protein
3E: UL31
3F: UL34 protein
3G: UL31
3H: UL34 protein
3I: UL31
3J: UL34 protein
3K: UL31
3L: UL34 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,042,224168
Polymers2,037,98884
Non-polymers4,23684
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
UL31 / UL31 protein


Mass: 28235.549 Da / Num. of mol.: 42
Source method: isolated from a genetically manipulated source
Details: Two proteins (PUL31 AND PUL34) of pseudorabies virus (PRV) are co-expressed in the cells forming there the herpesviral nuclear egress complex lining as a coat perinuclear vesicles.
Source: (gene. exp.) Suid herpesvirus 1 / Gene: UL31 / Plasmid: pETDUET / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): LEMO21 / References: UniProt: G3G955
#2: Protein ...
UL34 protein


Mass: 20287.975 Da / Num. of mol.: 42
Source method: isolated from a genetically manipulated source
Details: Two proteins (PUL31 AND PUL34) of pseudorabies virus (PRV) are co-expressed in the cells forming there the herpesviral nuclear egress complex lining as a coat perinuclear vesicles.
Source: (gene. exp.) Suid herpesvirus 1 / Gene: UL34 / Plasmid: pETDUET / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): LEMO21 / References: UniProt: G3G8R3
#3: Chemical...
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 42 / Source method: obtained synthetically / Formula: Zn
#4: Chemical...
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 42 / Source method: obtained synthetically / Formula: Cl

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: electron tomography

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Sample preparation

ComponentName: PORCINE EPITHELIAL-LIKE EMBRYONIC EFN-R KIDNEY CELLS STABLY CO- EXPRESSING PRV UL31 AND UL34, THE LATTER FUSED WITH GFP (CELL LINE DESIGNATED AS BK-EF-UL31- 34GFP CATALOGUE NO. RIE 1083 OF THE ...Name: PORCINE EPITHELIAL-LIKE EMBRYONIC EFN-R KIDNEY CELLS STABLY CO- EXPRESSING PRV UL31 AND UL34, THE LATTER FUSED WITH GFP (CELL LINE DESIGNATED AS BK-EF-UL31- 34GFP CATALOGUE NO. RIE 1083 OF THE COLLECTION OF CELL LINES IN VETERINARY MEDICINE AT THE FLI, GREIFSWALD-INSEL RIEMS, GERMANY)
Type: CELL
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO
Specimen supportDetails: OTHER
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE-PROPANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE-PROPANE MIXTURE, INSTRUMENT- HOMEMADE PLUNGER, METHOD- BLOTTED MANUALLY WITH A BENT STRIP OF WHATMAN NO. 1 FILTER PAPER FROM THE NON-COATED GRID SIDE FOR 2 ...Details: VITRIFICATION 1 -- CRYOGEN- ETHANE-PROPANE MIXTURE, INSTRUMENT- HOMEMADE PLUNGER, METHOD- BLOTTED MANUALLY WITH A BENT STRIP OF WHATMAN NO. 1 FILTER PAPER FROM THE NON-COATED GRID SIDE FOR 2 TO 3 S IMMEDIATELY BEFORE VITRIFICATION BY THE GRAVITY-DRIVEN PLUNGING APPARATUS IN A ETHANE-PROPANE MIXTURE COOLED BY LIQUID NITROGEN, TIMERESOLVEDSTATE- TWO DAYS OF INCUBATION (37 DEGREE C, 5 PERCENT CO2) IN PLASTIC MICROSCOPE SLIDE GROWTH CHAMBERS (MUE-SLIDE 2X9 WELL, IBIDI GMBH) BEFORE CRYO- IMMOBILIZATION,

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Apr 26, 2013 / Details: 2048 X 2048
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Calibrated magnification: 52650 X / Nominal defocus max: 6000 nm / Nominal defocus min: -6000 nm / Cs: 2 mm
Specimen holderTilt angle max: 52 ° / Tilt angle min: -50 °
Image recordingElectron dose: 114 e/Å2 / Film or detector model: GATAN MULTISCAN
Image scansNum. digital images: 35

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Processing

EM software
IDNameCategory
1TEMPymodel fitting
2IMOD3D reconstruction
3PEET3D reconstruction
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionMethod: WEIGHTED BACK PROJECTION / Resolution: 35 Å / Num. of particles: 300 / Actual pixel size: 11.4 Å
Details: FIT OF HEXAMERIC LATTICE OF NEC INTO SUBTOMOGRAM AVERAGE. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3197.
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Target criteria: MINIMISATION OF ATOMIC CLASHES AND PROTRUSION FROM MAP
Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 5E8C

5e8c

RefinementHighest resolution: 35 Å
Refinement stepCycle: LAST / Highest resolution: 35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms108024 0 84 0 108108

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