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- EMDB-29451: Tracing p2 phiPA3 PhuN Tetramer Interfaces -

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Basic information

Entry
Database: EMDB / ID: EMD-29451
TitleTracing p2 phiPA3 PhuN Tetramer Interfaces
Map dataCore PhuN tetramer processed with larger mask and an interior mask to better resolve contacts made between tetramers.
Sample
  • Complex: Core tetramer assembly (p2) of the phiPA3 bacteriophage PhuN protein with neighboring subunits
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein, phiPA3 PhuN
KeywordsphiPA3 protein / shell protein / PhuN / phage nucleus / VIRAL PROTEIN
Function / homology
Function and homology information


detection of maltose stimulus / maltose transport complex / carbohydrate transport / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex / cell chemotaxis ...detection of maltose stimulus / maltose transport complex / carbohydrate transport / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / outer membrane-bounded periplasmic space / host cell cytoplasm / periplasmic space / DNA damage response / membrane
Similarity search - Function
Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
Chimallin / Maltose/maltodextrin-binding periplasmic protein
Similarity search - Component
Biological speciesPseudomonas phage PhiPA3 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.21 Å
AuthorsNieweglowska ES / Brilot AF / Mendez-Moran M / Kokontis C / Baek M / Li J / Cheng Y / Baker D / Bondy-Denomy J / Agard DA
Funding support United States, 2 items
OrganizationGrant numberCountry
Department of Health & Human Services (HHS)R35GM118099 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Commun / Year: 2023
Title: The ϕPA3 phage nucleus is enclosed by a self-assembling 2D crystalline lattice.
Authors: Eliza S Nieweglowska / Axel F Brilot / Melissa Méndez-Moran / Claire Kokontis / Minkyung Baek / Junrui Li / Yifan Cheng / David Baker / Joseph Bondy-Denomy / David A Agard /
Abstract: To protect themselves from host attack, numerous jumbo bacteriophages establish a phage nucleus-a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host ...To protect themselves from host attack, numerous jumbo bacteriophages establish a phage nucleus-a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the phage nucleus while other phage and host proteins are excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that nucleus fragments isolated from ϕPA3 infected Pseudomonas aeruginosa form a 2-dimensional lattice, having p2 or p4 symmetry. We further demonstrate that recombinantly purified primary Phage Nuclear Enclosure (PhuN) protein spontaneously assembles into similar 2D sheets with p2 and p4 symmetry. We resolve the dominant p2 symmetric state to 3.9 Å by cryo-EM. Our structure reveals a two-domain core, organized into quasi-symmetric tetramers. Flexible loops and termini mediate adaptable inter-tetramer contacts that drive subunit assembly into a lattice and enable the adoption of different symmetric states. While the interfaces between subunits are mostly well packed, two are open, forming channels that likely have functional implications for the transport of proteins, mRNA, and small molecules.
History
DepositionJan 14, 2023-
Header (metadata) releaseMar 1, 2023-
Map releaseMar 1, 2023-
UpdateSep 6, 2023-
Current statusSep 6, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29451.png

Downloads & links

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Map

FileDownload / File: emd_29451.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCore PhuN tetramer processed with larger mask and an interior mask to better resolve contacts made between tetramers.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 512 pix.
= 427.008 Å		0.83 Å/pix.
x 512 pix.
= 427.008 Å		0.83 Å/pix.
x 512 pix.
= 427.008 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.325
Minimum - Maximum-2.3450756 - 4.0027924
Average (Standard dev.)-0.0099512385 (±0.15693985)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 427.008 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_29451_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29451_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Core tetramer assembly (p2) of the phiPA3 bacteriophage PhuN prot...

EntireName: Core tetramer assembly (p2) of the phiPA3 bacteriophage PhuN protein with neighboring subunits
Components
  • Complex: Core tetramer assembly (p2) of the phiPA3 bacteriophage PhuN protein with neighboring subunits
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein, phiPA3 PhuN

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Supramolecule #1: Core tetramer assembly (p2) of the phiPA3 bacteriophage PhuN prot...

SupramoleculeName: Core tetramer assembly (p2) of the phiPA3 bacteriophage PhuN protein with neighboring subunits
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas phage PhiPA3 (virus)

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Macromolecule #1: Maltose/maltodextrin-binding periplasmic protein, phiPA3 PhuN

MacromoleculeName: Maltose/maltodextrin-binding periplasmic protein, phiPA3 PhuN
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage PhiPA3 (virus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: HHHHHHMKIE EGKLVIWING DKGYNGLAEV GKKFEKDTGI KVTVEHPDKL EEKFPQVAAT GDGPDIIFWA HDRFGGYAQS GLLAEITPDK AFQDKLYPFT WDAVRYNGKL IAYPIAVEAL SLIYNKDLLP NPPKTWEEIP ALDKELKAKG KSALMFNLQE PYFTWPLIAA ...String:
HHHHHHMKIE EGKLVIWING DKGYNGLAEV GKKFEKDTGI KVTVEHPDKL EEKFPQVAAT GDGPDIIFWA HDRFGGYAQS GLLAEITPDK AFQDKLYPFT WDAVRYNGKL IAYPIAVEAL SLIYNKDLLP NPPKTWEEIP ALDKELKAKG KSALMFNLQE PYFTWPLIAA DGGYAFKYEN GKYDIKDVGV DNAGAKAGLT FLVDLIKNKH MNADTDYSIA EAAFNKGETA MTINGPWAWS NIDTSKVNYG VTVLPTFKGQ PSKPFVGVLS AGINAASPNK ELAKEFLENY LLTDEGLEAV NKDKPLGAVA LKSYEEELAK DPRIAATMEN AQKGEIMPNI PQMSAFWYAV RTAVINAASG RQTVDEALKD AQTGKPIPNP LLGLDSTENL YFQGMQQTQQ GPKVQTQTLQ GGAGNLNSIF QRSGRTDGGD ARASEALAVF NKLKEEAIAQ QDLHDDFLVF RFDRDQNRVG YSALLVVKRA AINGQQVIVT RPLVMPNDQI TLPTKKLTIQ NGMHQETIEA EADVQDVFTT QYWNRICDSI RQQTGKHDAM VINAGPTVIP ADFDLKDELV LKQLLIKSVN LCDDMLAKRS GEQPFSVAML KGTDETLAAR LNFTGKPMHD SLGYPIRSDI LVSLNRVKKP GQQENEFYEA EDKLNQVSCF VNLEYTPQPQ QAIYGAPQQT QQLPPLTPAI VITDVRQAEW LKANTMELYL FALSNAFRVT ANQSWARSLL PQLGKVKDMR DIGAIGYLSR LAARVETKTE TFTDQNFAEL LYNMVRPSPV FMSDLNRFGD NAAIENVFID ALGGVNQQRA VAAIIAGVNN LIGGGFEKFF DHNTMPIIQP YGTDIQLGYY LDGEGEKQDR RDLDVLGALN ASDGNIQEWM SWYGTQCNVA VHPELRARQS KNFDRQYLGN SVTYTTRAHR GIWNPKFIEA LDKAIASVGL TVAMDNVAQV FGAQRFSGNL AIADYAVTGT AQVSSGLVSN GGYNPQFGVG QGSGFY

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

BufferpH: 6.5
Component:
ConcentrationName
20.0 mMBisTrisPropane
150.0 mMSodium Chloride
1.0 mMDTT
1.0 mMEDTA
2.0 mMMagnesium Chloride
1.0 mMATP

Details: 0.25 Complete Protease Inhibitor Tablet also included
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 67.0 e/Å2
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: 0.8 µm / Nominal magnification: 105000
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

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.21 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0-beta) / Number images used: 161270
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v2.15.0) / Software - details: Ab-Initio Reconstruction
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 1.0.0-beta)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. v3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8fv5:
Representation of 16-mer phiPA3 PhuN Lattice, p2

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