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Entry
Database: PDB / ID: 5muv
TitleAtomic structure fitted into a localized reconstruction of bacteriophage phi6 packaging hexamer P4
ComponentsPackaging enzyme P4
KeywordsHYDROLASE / packaging / ATPase / vertex / hyrdolase
Function / homology
Function and homology information


viral procapsid / viral genome packaging / ribonucleoside triphosphate phosphatase activity / viral capsid / nucleoside-triphosphate phosphatase / ATP binding
Similarity search - Function
Packaging enzyme P4 / ATPase P4 of dsRNA bacteriophage phi-12 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Packaging enzyme P4
Similarity search - Component
Biological speciesPseudomonas phage phi6 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.1 Å
AuthorsSun, Z. / El Omari, K. / Sun, X. / Ilca, S. / Kotecha, A. / Stuart, D.I. / Poranen, M.M. / Huiskonen, J.T.
Funding support Finland, 1items
OrganizationGrant numberCountry
Finland
Citation
Journal: Nat Commun / Year: 2017
Title: Double-stranded RNA virus outer shell assembly by bona fide domain-swapping.
Authors: Zhaoyang Sun / Kamel El Omari / Xiaoyu Sun / Serban L Ilca / Abhay Kotecha / David I Stuart / Minna M Poranen / Juha T Huiskonen /
Abstract: Correct outer protein shell assembly is a prerequisite for virion infectivity in many multi-shelled dsRNA viruses. In the prototypic dsRNA bacteriophage φ6, the assembly reaction is promoted by ...Correct outer protein shell assembly is a prerequisite for virion infectivity in many multi-shelled dsRNA viruses. In the prototypic dsRNA bacteriophage φ6, the assembly reaction is promoted by calcium ions but its biomechanics remain poorly understood. Here, we describe the near-atomic resolution structure of the φ6 double-shelled particle. The outer T=13 shell protein P8 consists of two alpha-helical domains joined by a linker, which allows the trimer to adopt either a closed or an open conformation. The trimers in an open conformation swap domains with each other. Our observations allow us to propose a mechanistic model for calcium concentration regulated outer shell assembly. Furthermore, the structure provides a prime exemplar of bona fide domain-swapping. This leads us to extend the theory of domain-swapping from the level of monomeric subunits and multimers to closed spherical shells, and to hypothesize a mechanism by which closed protein shells may arise in evolution.
#1: Journal: Nucleic Acids Res / Year: 2013
Title: Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution.
Authors: Kamel El Omari / Christoph Meier / Denis Kainov / Geoff Sutton / Jonathan M Grimes / Minna M Poranen / Dennis H Bamford / Roman Tuma / David I Stuart / Erika J Mancini /
Abstract: Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, ...Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, 12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from 6, 8 and 13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in 8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in 12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the 12 enzyme.
History
DepositionJan 14, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 22, 2017Provider: repository / Type: Initial release
Revision 2.0Aug 2, 2017Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Experimental preparation / Refinement description
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / em_3d_fitting / em_sample_support / em_software / pdbx_struct_conn_angle / pdbx_validate_symm_contact / struct_conn / struct_conn_type / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _atom_site_anisotrop.U[1][1] / _atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] / _atom_site_anisotrop.U[2][2] / _atom_site_anisotrop.U[2][3] / _atom_site_anisotrop.U[3][3] / _atom_site_anisotrop.pdbx_auth_asym_id / _atom_site_anisotrop.pdbx_auth_atom_id / _atom_site_anisotrop.pdbx_auth_comp_id / _atom_site_anisotrop.pdbx_auth_seq_id / _atom_site_anisotrop.pdbx_label_asym_id / _atom_site_anisotrop.pdbx_label_atom_id / _atom_site_anisotrop.pdbx_label_comp_id / _atom_site_anisotrop.type_symbol / _em_3d_fitting.target_criteria / _em_sample_support.grid_type / _em_software.name / _struct_conn_type.id / _struct_site.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_seq_id / _struct_site_gen.auth_asym_id / _struct_site_gen.auth_seq_id / _struct_site_gen.label_asym_id
Revision 2.1Jan 31, 2018Group: Advisory / Data processing / Other
Category: cell / em_software ...cell / em_software / pdbx_validate_close_contact / pdbx_validate_symm_contact
Item: _cell.Z_PDB / _cell.angle_alpha ..._cell.Z_PDB / _cell.angle_alpha / _cell.angle_beta / _cell.angle_gamma / _cell.length_a / _cell.length_b / _cell.length_c / _em_software.details / _em_software.name
Revision 3.0Apr 24, 2019Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Other
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / atom_sites / em_admin / pdbx_database_proc / pdbx_nonpoly_scheme / struct_asym / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.pdbx_auth_asym_id / _atom_site.pdbx_auth_atom_name / _atom_site.pdbx_auth_comp_id / _atom_site.pdbx_auth_seq_id / _atom_site.type_symbol / _atom_site_anisotrop.U[1][1] / _atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] / _atom_site_anisotrop.U[2][2] / _atom_site_anisotrop.U[2][3] / _atom_site_anisotrop.U[3][3] / _atom_site_anisotrop.pdbx_PDB_atom_name / _atom_site_anisotrop.pdbx_PDB_residue_name / _atom_site_anisotrop.pdbx_PDB_residue_no / _atom_site_anisotrop.pdbx_PDB_strand_id / _atom_site_anisotrop.pdbx_auth_asym_id / _atom_site_anisotrop.pdbx_auth_atom_id / _atom_site_anisotrop.pdbx_auth_comp_id / _atom_site_anisotrop.pdbx_auth_seq_id / _atom_site_anisotrop.pdbx_label_asym_id / _atom_site_anisotrop.pdbx_label_atom_id / _atom_site_anisotrop.pdbx_label_comp_id / _atom_site_anisotrop.type_symbol / _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[1][2] / _atom_sites.fract_transf_matrix[1][3] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[2][3] / _atom_sites.fract_transf_matrix[3][3] / _em_admin.last_update / _pdbx_nonpoly_scheme.auth_mon_id / _pdbx_nonpoly_scheme.auth_seq_num / _pdbx_nonpoly_scheme.entity_id / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id / _pdbx_nonpoly_scheme.pdb_seq_num / _pdbx_nonpoly_scheme.pdb_strand_id / _struct_asym.entity_id / _struct_asym.pdbx_PDB_id / _struct_asym.pdbx_alt_id / _struct_asym.pdbx_type / _struct_site.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_seq_id / _struct_site_gen.auth_asym_id / _struct_site_gen.auth_seq_id / _struct_site_gen.label_asym_id

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Structure visualization

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Assembly

Deposited unit
A: Packaging enzyme P4
B: Packaging enzyme P4
I: Packaging enzyme P4
J: Packaging enzyme P4
K: Packaging enzyme P4
L: Packaging enzyme P4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)198,87618
Polymers196,0726
Non-polymers2,80412
Water0
1


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

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Components

#1: Protein
Packaging enzyme P4


Mass: 32678.740 Da / Num. of mol.: 6 / Fragment: RESIDUES 1-309
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage phi6 (bacteriophage) / Gene: P4 / Production host: Escherichia coli (E. coli)
References: UniProt: P11125, nucleoside-triphosphate phosphatase
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Pseudomonas phage phi6Pseudomonas virus phi6 / Type: VIRUS / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Pseudomonas phage phi6 (bacteriophage)
Details of virusEmpty: NO / Enveloped: YES / Isolate: SPECIES / Type: VIRION
Natural hostOrganism: Pseudomonas syringae
Buffer solutionpH: 7.2
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 37037 X / Calibrated defocus min: 300 nm / Calibrated defocus max: 3000 nm / Cs: 2 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER / Temperature (max): 120 K / Temperature (min): 80 K
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 0.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 900
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansSampling size: 5 µm / Width: 3710 / Height: 3710 / Movie frames/image: 22 / Used frames/image: 1-22

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Processing

EM software
IDNameVersionCategoryDetails
1ETHAN1.2particle selection
2SerialEMimage acquisition
4CTFFIND3CTF correction
5RELION1.4CTF correction
8UCSF Chimera1.12model fitting
101.1.0initial Euler assignmentLocalizedReconstruction
12RELION1.4classification
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 159492
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 9.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56448 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient

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