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- EMDB-29550: phiPA3 PhuN Tetramer, p2 -

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Basic information

Entry
Database: EMDB / ID: EMD-29550
TitlephiPA3 PhuN Tetramer, p2
Map data
Sample
  • Complex: Core tetramer assembly (p2) of the phiPA3 bacteriophage PhuN protein
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein, phiPA3 PhuN
Biological speciesPseudomonas phage PhiPA3 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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 25, 2023-
Header (metadata) releaseMar 1, 2023-
Map releaseMar 1, 2023-
UpdateMar 1, 2023-
Current statusMar 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29550.png

Downloads & links

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Map

FileDownload / File: emd_29550.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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

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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: 0.7
Minimum - Maximum-4.272476 - 7.6364574
Average (Standard dev.)-0.012481492 (±0.21606901)
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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Mask #1

Fileemd_29550_msk_1.map
Projections & Slices
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Half map: #2

Fileemd_29550_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29550_half_map_2.map
Projections & Slices
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Sample components

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

EntireName: Core tetramer assembly (p2) of the phiPA3 bacteriophage PhuN protein
Components
  • Complex: Core tetramer assembly (p2) of the phiPA3 bacteriophage PhuN protein
    • 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 protein

SupramoleculeName: Core tetramer assembly (p2) of the phiPA3 bacteriophage PhuN protein
type: complex / ID: 1 / Chimera: Yes / 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 GLLAEITPD KAFQDKLYPF TWDAVRYNGK LIAYPIAVEA LSLIYNKDLL PNPPKTWEEI PALDKELKAK GKSALMFNLQ E PYFTWPLI ...String:
HHHHHHMKIE EGKLVIWING DKGYNGLAEV GKKFEKDTGI KVTVEHPDKL EEKFPQVAAT GDGPDIIFWA HDRFGGYAQS GLLAEITPD KAFQDKLYPF TWDAVRYNGK LIAYPIAVEA LSLIYNKDLL PNPPKTWEEI PALDKELKAK GKSALMFNLQ E PYFTWPLI AADGGYAFKY ENGKYDIKDV GVDNAGAKAG LTFLVDLIKN KHMNADTDYS IAEAAFNKGE TAMTINGPWA WS NIDTSKV NYGVTVLPTF KGQPSKPFVG VLSAGINAAS PNKELAKEFL ENYLLTDEGL EAVNKDKPLG AVALKSYEEE LAK DPRIAA TMENAQKGEI MPNIPQMSAF WYAVRTAVIN AASGRQTVDE ALKDAQTGKP IPNPLLGLDS TENLYFQGMQ QTQQ GPKVQ TQTLQGGAGN LNSIFQRSGR TDGGDARASE ALAVFNKLKE EAIAQQDLHD DFLVFRFDRD QNRVGYSALL VVKRA AING QQVIVTRPLV MPNDQITLPT KKLTIQNGMH QETIEAEADV QDVFTTQYWN RICDSIRQQT GKHDAMVINA GPTVIP ADF DLKDELVLKQ LLIKSVNLCD DMLAKRSGEQ PFSVAMLKGT DETLAARLNF TGKPMHDSLG YPIRSDILVS LNRVKKP GQ QENEFYEAED KLNQVSCFVN LEYTPQPQQA IYGAPQQTQQ LPPLTPAIVI TDVRQAEWLK ANTMELYLFA LSNAFRVT A NQSWARSLLP QLGKVKDMRD IGAIGYLSRL AARVETKTET FTDQNFAELL YNMVRPSPVF MSDLNRFGDN AAIENVFID ALGGVNQQRA VAAIIAGVNN LIGGGFEKFF DHNTMPIIQP YGTDIQLGYY LDGEGEKQDR RDLDVLGALN ASDGNIQEWM SWYGTQCNV AVHPELRARQ SKNFDRQYLG NSVTYTTRAH RGIWNPKFIE ALDKAIASVG LTVAMDNVAQ VFGAQRFSGN L AIADYAVT GTAQVSSGLV SNGGYNPQFG VGQGSGFY

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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

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 29303
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: 4 / Software - Name: cisTEM (ver. 1.0.0-beta)
FSC plot (resolution estimation)

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