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- PDB-7sxk: Kinetically trapped Pseudomonas-phage PaP3 portal protein - Full ... -

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Basic information

Entry
Database: PDB / ID: 7sxk
TitleKinetically trapped Pseudomonas-phage PaP3 portal protein - Full Length
ComponentsPortal protein
KeywordsVIRAL PROTEIN / portal protein / dodecamer
Function / homologyORF.04
Function and homology information
Biological speciesPseudomonas virus PaP3
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsHou, C.F.D. / Swanson, N.A. / Li, F. / Yang, R. / Lokareddy, R.K. / Cingolani, G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 GM100888, R35 GM140733-0, P30 CA56036, HSSN261200800001E United States
CitationJournal: J Mol Biol / Year: 2022
Title: Cryo-EM Structure of a Kinetically Trapped Dodecameric Portal Protein from the Pseudomonas-phage PaP3.
Authors: Chun-Feng David Hou / Nicholas A Swanson / Fenglin Li / Ruoyu Yang / Ravi K Lokareddy / Gino Cingolani /
Abstract: Portal proteins are dodecameric assemblies that occupy a unique 5-fold vertex of the icosahedral capsid of tailed bacteriophages and herpesviruses. The portal vertex interrupts the icosahedral ...Portal proteins are dodecameric assemblies that occupy a unique 5-fold vertex of the icosahedral capsid of tailed bacteriophages and herpesviruses. The portal vertex interrupts the icosahedral symmetry, and in vivo, its assembly and incorporation in procapsid are controlled by the scaffolding protein. Ectopically expressed portal oligomers are polymorphic in solution, and portal rings built by a different number of subunits have been documented in the literature. In this paper, we describe the cryo-EM structure of the portal protein from the Pseudomonas-phage PaP3, which we determined at 3.4 Å resolution. Structural analysis revealed a dodecamer with helical rather than rotational symmetry, which we hypothesize is kinetically trapped. The helical assembly was stabilized by local mispairing of portal subunits caused by the slippage of crown and barrel helices that move like a lever with respect to the portal body. Removing the C-terminal barrel promoted assembly of undecameric and dodecameric rings with quasi-rotational symmetry, suggesting that the barrel contributes to subunits mispairing. However, ΔC-portal rings were intrinsically asymmetric, with most particles having one open portal subunit interface. Together, these data expand the structural repertoire of viral portal proteins to Pseudomonas-phages and shed light on the unexpected plasticity of the portal protein quaternary structure.
History
DepositionNov 23, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 20, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
b: Portal protein
a: Portal protein
l: Portal protein
k: Portal protein
j: Portal protein
i: Portal protein
h: Portal protein
c: Portal protein
d: Portal protein
e: Portal protein
f: Portal protein
g: Portal protein


Theoretical massNumber of molelcules
Total (without water)971,77612
Polymers971,77612
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
2
b: Portal protein
a: Portal protein
l: Portal protein
k: Portal protein
j: Portal protein
i: Portal protein
h: Portal protein
c: Portal protein
d: Portal protein
e: Portal protein
f: Portal protein


Theoretical massNumber of molelcules
Total (without water)890,79511
Polymers890,79511
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area95810 Å2
ΔGint-453 kcal/mol
Surface area291530 Å2

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Components

#1: Protein
Portal protein


Mass: 80981.344 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas virus PaP3 / Gene: orf4 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8H9R8

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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: Asymmetric dodecamer complex of phage PaP3 portal / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Pseudomonas virus PaP3
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
120 mMTris-HClTris1
2200 mMNaClSodium chloride1
35 mMEDTAEthylenediaminetetraacetic acid1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: CTFFIND / Version: 4 / Category: CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2500000
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 28000 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00349718
ELECTRON MICROSCOPYf_angle_d0.69367380
ELECTRON MICROSCOPYf_dihedral_angle_d6.377016
ELECTRON MICROSCOPYf_chiral_restr0.0447577
ELECTRON MICROSCOPYf_plane_restr0.0048945

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