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- EMDB-43103: Structure of the PARIS immune complex with AriB subunits in C3 ar... -

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Basic information

Entry
Database: EMDB / ID: EMD-43103
TitleStructure of the PARIS immune complex with AriB subunits in C3 arrangement.
Map dataSharpened map of final reconstruction.
Sample
  • Complex: PARIS immune complex in the assembled state with AriB subunits in a C3-symmetric arrangement.
    • Protein or peptide: AriA, ABC ATPase and sensor of PARIS immunity
    • Protein or peptide: AriB, DUF4435 and TOPRIM nuclease. Effector of the PARIS immune complex.
KeywordsPARIS / AriA / AriB / DUF4435 / IMMUNE SYSTEM
Biological speciesEscherichia coli B185 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsBurman NB / Henriques W / Wilkinson R / Graham A / Wiedenheft B
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM134867 United States
CitationJournal: To Be Published
Title: Activation of the PARIS immune complex by viral proteins results in host tRNA cleavage and can be overcome by viruses encoding non-cleavable tRNAs
Authors: Burman NB / Belukhina S / Depardieu F / Wilkinson R / Skutel M / Santiago-Frangos A / Graham A / Livenskyi A / Chechenina A / Morozova N / Zahl T / Henriques W / Buyukyoruk M / Rouillon C / ...Authors: Burman NB / Belukhina S / Depardieu F / Wilkinson R / Skutel M / Santiago-Frangos A / Graham A / Livenskyi A / Chechenina A / Morozova N / Zahl T / Henriques W / Buyukyoruk M / Rouillon C / Shyrokova O / Suzuki T / Hauryliuk V / Severinov K / Groseille J / Thierry A / Koszul R / Tesson F / Bernheim A / Bikard D / Wiedenheft B / Isaev A
History
DepositionDec 11, 2023-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43103.png

Downloads & links

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Map

FileDownload / File: emd_43103.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of final reconstruction.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 300 pix.
= 331.2 Å		1.1 Å/pix.
x 300 pix.
= 331.2 Å		1.1 Å/pix.
x 300 pix.
= 331.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.104 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.00083379634 - 2.0434291
Average (Standard dev.)0.0029062042 (±0.038592033)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 331.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map A used for sharpening

Fileemd_43103_half_map_1.map
AnnotationHalf Map A used for sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B used for sharpening

Fileemd_43103_half_map_2.map
AnnotationHalf Map B used for sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PARIS immune complex in the assembled state with AriB subunits in...

EntireName: PARIS immune complex in the assembled state with AriB subunits in a C3-symmetric arrangement.
Components
  • Complex: PARIS immune complex in the assembled state with AriB subunits in a C3-symmetric arrangement.
    • Protein or peptide: AriA, ABC ATPase and sensor of PARIS immunity
    • Protein or peptide: AriB, DUF4435 and TOPRIM nuclease. Effector of the PARIS immune complex.

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Supramolecule #1: PARIS immune complex in the assembled state with AriB subunits in...

SupramoleculeName: PARIS immune complex in the assembled state with AriB subunits in a C3-symmetric arrangement.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli B185 (bacteria)

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Macromolecule #1: AriA, ABC ATPase and sensor of PARIS immunity

MacromoleculeName: AriA, ABC ATPase and sensor of PARIS immunity / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli B185 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAIRTISKIE LSKIHNRYNL TVDFFNDLNV IHGKNGAGKS TLIHVIANIV NGDFIRFAFL IFEEIKATYS DGLKIVIRRD KIDEQSFISV TLSNGKYIKF AVGEAMATVR EIESERHLRE RDVKSMLAMD IDKFVKENEL QKVRASYFPA FRTMLEAWSS SSDVGYERRV ...String:
MAIRTISKIE LSKIHNRYNL TVDFFNDLNV IHGKNGAGKS TLIHVIANIV NGDFIRFAFL IFEEIKATYS DGLKIVIRRD KIDEQSFISV TLSNGKYIKF AVGEAMATVR EIESERHLRE RDVKSMLAMD IDKFVKENEL QKVRASYFPA FRTMLEAWSS SSDVGYERRV IRSSFYNRKA SAFARELFGQ FLPSINYPSP MEIEDRLREE IRRAQLGIAA YESRTFSESF VKVFSALFDN SSVEGEITGE LLKEIEGLAI AQDSSIKNGY YAEYSKVYEE IRSLINRNLK GKVENSVSGA LVVYRDALRD RQDYQEKAFS EIDNYMSSVN SFLEDKEMAY DFDLRRKYPK VGLKFPDGSW SPIRVLSSGE RQLLTMLYAA SKMGDDAIVL IDEPEISLHI DWQEDLLKRM LSQLSGRQII VCTHSPSIAT GYEDFMINIS PEFISSRDND NHKDSEEMEE DESL

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Macromolecule #2: AriB, DUF4435 and TOPRIM nuclease. Effector of the PARIS immune c...

MacromoleculeName: AriB, DUF4435 and TOPRIM nuclease. Effector of the PARIS immune complex.
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli B185 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSSCAYTIDS YITLLTMSSK KRLLVEGRHD RSHLYQLIYK FNPASKVKID TAQDIKASDK AMSKNNRLKI ETIHSKVKGK DNISFLCDRE FREFAFNDQI EDLLNSHYCD DSLYWTLGHS LENYFFNPSI IIDAFQFLSP SEYKYKAIEL FSELISSSFA VLAAVSLAAK ...String:
MSSCAYTIDS YITLLTMSSK KRLLVEGRHD RSHLYQLIYK FNPASKVKID TAQDIKASDK AMSKNNRLKI ETIHSKVKGK DNISFLCDRE FREFAFNDQI EDLLNSHYCD DSLYWTLGHS LENYFFNPSI IIDAFQFLSP SEYKYKAIEL FSELISSSFA VLAAVSLAAK DIDKAGLPAA LIDWKDIVIN DGTIKLIRRD SYDIDSACVD SFFNAFDAVL PRVIASDVGI CSRVVRGHTG ILLLQKLFSA CLYYVGREDD ALQADSSANY FCNLSELSLT TALAESWVRK IGVLEDVYFP DSLLKNIEWS HPQFEK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7340 / Average electron dose: 56.38 e/Å2
Electron beamAcceleration voltage: 200 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: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4078384
Details: Particles Extracted for initial classification Template for particle picking generated from processing a 200 micrograph subset of the data using the blob picker. A volume corresponding to ...Details: Particles Extracted for initial classification Template for particle picking generated from processing a 200 micrograph subset of the data using the blob picker. A volume corresponding to the assembled PARIS complex was identified and Alphafold2 models were docked into the map. ChimeraX's molmap command was used to generate a 20 Angstrom lowpass filtered volume of the docked Alphafold2 models and imported to Cryosparc for template generation.
Startup modelType of model: NONE
Details: Multiclass ab-initio reconstructions were used to sort particle images and obtain a consensus refinement.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.41) / Software - details: Non-Uniform Refinement
Details: cryoSPARC's implementation of Gold-Standard FSC calculation was used.
Number images used: 197018
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.41) / Software - details: Ab-Initio Reconstruction
Details: A three class ab-initio reconstruction was used to remove junk classes.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.41) / Software - details: Non-Uniform Refinement
Details: After multiple rounds of Ab-initio reconstruction and heterogenous refinement, a 532,010 particle stack was isolated that corresponded to the fully assembled PARIS complex. In this volume, ...Details: After multiple rounds of Ab-initio reconstruction and heterogenous refinement, a 532,010 particle stack was isolated that corresponded to the fully assembled PARIS complex. In this volume, one asymmetric unit of the complex is well-aligned, while in the others, the AriB subunits are present in two, mutually exclusive orientations. This mixture of particles in the assembled PARIS complex correspond to the Cis and Trans arrangements of the complex and as such, the particles were further sorted using focused 3-D classification to isolate the two structural isomers of the complex.
Final 3D classificationNumber classes: 2 / Avg.num./class: 211318 / Software - Name: cryoSPARC (ver. 4.41) / Software - details: Focused 3-D Classification
Details: From the 532,010 particle stack identified above, multiple rounds of focused 3-D Classification were used to isolate particle images of the PARIS complex with AriB subunits in the C3 arrangement.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8ux9


Chain - Source name: PDB / Chain - Initial model type: experimental model
Details: The initial model consisted of 3 copies of the PDB entry 8UX9
DetailsRigid body fitting was done using the fitmap command in ChimeraX to dock 3 copies of the experimentally determined structure of the asymmetric unit into the density map of the fully assembled complex.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 158.6 / Target criteria: Cross-correlation coefficient

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