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- EMDB-12885: Structure of the apo-state of the bacteriophage PhiKZ non-virion ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12885
TitleStructure of the apo-state of the bacteriophage PhiKZ non-virion RNA polymerase - class including clamp
Map dataPhiKZ non-virion RNA polymerase - class including clamp
Sample
  • Complex: Bacteriophage PhiKZ non-virion RNA polymerase
    • Protein or peptide: PHIKZ055
    • Protein or peptide: PHIKZ068
    • Protein or peptide: DNA-directed RNA polymerasePolymerase
    • Protein or peptide: PHIKZ074
    • Protein or peptide: PHIKZ123
  • Ligand: ZINC ION
KeywordsRNA polymerase / PhiKZ / non-virion / TRANSCRIPTION
Function / homologyPHIKZ123 / PHIKZ074 / PHIKZ068 / PHIKZ055
Function and homology information
Biological speciesPseudomonas virus phiKZ / Pseudomonas phage phiKZ (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
Authorsde Martin Garrido N / Lai Wan Loong YTE
Funding support United Kingdom, Russian Federation, 2 items
OrganizationGrant numberCountry
Wellcome Trust206212/Z/17/Z United Kingdom
Russian Science Foundation19-74-10030 Russian Federation
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Structure of the bacteriophage PhiKZ non-virion RNA polymerase.
Authors: Natàlia deYMartín Garrido / Mariia Orekhova / Yuen Ting Emilie Lai Wan Loong / Anna Litvinova / Kailash Ramlaul / Tatyana Artamonova / Alexei S Melnikov / Pavel Serdobintsev / Christopher ...Authors: Natàlia deYMartín Garrido / Mariia Orekhova / Yuen Ting Emilie Lai Wan Loong / Anna Litvinova / Kailash Ramlaul / Tatyana Artamonova / Alexei S Melnikov / Pavel Serdobintsev / Christopher H S Aylett / Maria Yakunina /
Abstract: Bacteriophage ΦKZ (PhiKZ) is the archetype of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, ...Bacteriophage ΦKZ (PhiKZ) is the archetype of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. ΦKZ is an incredibly interesting virus, expressing many systems that the host already possesses. On infection, it forms a 'nucleus', erecting a barrier around its genome to exclude host endonucleases and CRISPR-Cas systems. ΦKZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. ΦKZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial β/β' subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of ΦKZ nvRNAP to better than 3.0 Å, shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, the position, structural homology and biochemical role of which imply that it has evolved from an ancestral homologue to σ-factor.
History
DepositionMay 7, 2021-
Header (metadata) releaseJul 7, 2021-
Map releaseJul 7, 2021-
UpdateNov 22, 2023-
Current statusNov 22, 2023Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-7ogp
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12885.png

Downloads & links

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Map

FileDownload / File: emd_12885.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPhiKZ non-virion RNA polymerase - class including clamp
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.0606534 - 0.0910257
Average (Standard dev.)0.00036268804 (±0.004750127)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.850.850.85
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z217.600217.600217.600
α/β/γ90.00090.00090.000
start NX/NY/NZ192139186
NX/NY/NZ211274246
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0610.0910.000

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Supplemental data

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Half map: PhiKZ non-virion RNA polymerase - class including clamp - halfmap 1

Fileemd_12885_half_map_1.map
AnnotationPhiKZ non-virion RNA polymerase - class including clamp - halfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: PhiKZ non-virion RNA polymerase - class including clamp - halfmap 2

Fileemd_12885_half_map_2.map
AnnotationPhiKZ non-virion RNA polymerase - class including clamp - halfmap 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Bacteriophage PhiKZ non-virion RNA polymerase

EntireName: Bacteriophage PhiKZ non-virion RNA polymerase
Components
  • Complex: Bacteriophage PhiKZ non-virion RNA polymerase
    • Protein or peptide: PHIKZ055
    • Protein or peptide: PHIKZ068
    • Protein or peptide: DNA-directed RNA polymerasePolymerase
    • Protein or peptide: PHIKZ074
    • Protein or peptide: PHIKZ123
  • Ligand: ZINC ION

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Supramolecule #1: Bacteriophage PhiKZ non-virion RNA polymerase

SupramoleculeName: Bacteriophage PhiKZ non-virion RNA polymerase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Details: Expressed in E.coli from sequences obtained from the St. Petersburg lab strain of bacteriophage PhiKZ
Source (natural)Organism: Pseudomonas virus phiKZ / Strain: St. Petersburg
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: PHIKZ055

MacromoleculeName: PHIKZ055 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage phiKZ (virus)
Molecular weightTheoretical: 57.976605 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MGLYAKVVDH NEVHDQFTGK RIYANDYNTS NSDEKEEFDR HFYSHFQDSE AIESSVSCDC RAIEDAHKL GVICDICNTP VVNTSSRPIE PSMWVRTPKH VRSLINPRLI IMLTGYLVTK EFDFLAYLTD TSYRYDVESI G SKETRRKV ...String:
MGSSHHHHHH SSGLVPRGSH MGLYAKVVDH NEVHDQFTGK RIYANDYNTS NSDEKEEFDR HFYSHFQDSE AIESSVSCDC RAIEDAHKL GVICDICNTP VVNTSSRPIE PSMWVRTPKH VRSLINPRLI IMLTGYLVTK EFDFLAYLTD TSYRYDVESI G SKETRRKV DRLLHRGFER GLNHFIDNFN EIFQFLLDAN IISNNKSEFA QFVAQNKDKL FPKYLPVPSK LCFVAESTTS GT YLDKPIE AAIDATLTFA SIDASSVPLS PIKAQNRTMR GLRLYGQFYE IYAKSRIAQK PGLARRHMFG ARLNATARAV ITS ISDPHD YDELHIPWGV GCQLLKYHLT NKLKAKFNMT TREAFSFVYE NVLQYNQIIA DLFKELIAEA APYKGMGCTF HRNP TLQRG STQQFFITKV KDDINDNSIS MSVLCLKAPN ADFDGDQLNL TLMPDVYLTK ATERIAPHTW VLSIDEPHEI SGNLE LQGP VVETIINWAH EKYLPPLEEW LKAK

UniProtKB: PHIKZ055

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Macromolecule #2: PHIKZ068

MacromoleculeName: PHIKZ068 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage phiKZ (virus)
Molecular weightTheoretical: 59.41977 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEIIVTGVQG TGFTEVATEH NGKRLTWTTT AYSKIRVQDQ QRVFQEINDY WSGLSAEAQQ HIWNCYVEIR KIMDMAMDPM RIAMSLSYY IKEMYKAMPM NSFRRWLLTI GKLYIPVDIE EVITDDSRYN RPDQTYLKHD YINLASVSLA LRPLVPIWGE F IDQGTSQE ...String:
MEIIVTGVQG TGFTEVATEH NGKRLTWTTT AYSKIRVQDQ QRVFQEINDY WSGLSAEAQQ HIWNCYVEIR KIMDMAMDPM RIAMSLSYY IKEMYKAMPM NSFRRWLLTI GKLYIPVDIE EVITDDSRYN RPDQTYLKHD YINLASVSLA LRPLVPIWGE F IDQGTSQE MHKECEVISL ISDCEVNHWP VDEISIDGTP VETAYDKLSA YVKFCVEDEA PTLANLYRGM SSAEVPDILQ AK VMVRRLT ILPLNDATSH SIVSNMFRYV KSNLNPAERS TADRVNDKRP DKGGIDDDDK TSFIESHKTK QRVTPGDIVA YNL DALDVV KLVHKIDDTV PVELIQECLD CVAVTATKDI YPHQILLAQW VMHKAFPARA FSHINKNAVN HLLAAAQSLM WHWG FQQVA VFMQVELYYS GEHAMSIQPR NSTRIQIKYK DVMDELYPHQ RQQRAINGVP VAPVNIAGIA VQSAHASIRS SNWIY HGPD RLFKEAEQVT QNKVLVVPAT IKSVITELVI HLGKLNQ

UniProtKB: PHIKZ068

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Macromolecule #3: DNA-directed RNA polymerase

MacromoleculeName: DNA-directed RNA polymerase / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pseudomonas phage phiKZ (virus)
Molecular weightTheoretical: 78.780453 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSQLGRREID LTLLGHTGLD PWYGTTSSAR GAMFVTHIGQ APEVNGNESR YFLTGAELEY AKYTHDVRFP EDCRVLHVLR KYPTGIGKD SIRSNPVTTI IYENYFDKYK TIGVLHVPEY MSHHQDFGYE LVKNREVWET IAPNEMFSKD TVIAQSGAVK K DGTLGMGV ...String:
MSQLGRREID LTLLGHTGLD PWYGTTSSAR GAMFVTHIGQ APEVNGNESR YFLTGAELEY AKYTHDVRFP EDCRVLHVLR KYPTGIGKD SIRSNPVTTI IYENYFDKYK TIGVLHVPEY MSHHQDFGYE LVKNREVWET IAPNEMFSKD TVIAQSGAVK K DGTLGMGV NANVVFLSAA GTIEDGFVAN KNFLKRMMPT SYSTAVANAG RKAFFLNMYG DDKIYKPFPD IGDVIRPDGV IF AIRDHDD DLAPAEMTPR ALRTLDRTFD RAVIGTPGAK VIDIDIWRDE RVNPSPTPTG MDAQLVKYHT HLSSYYRELL KIY RGLLAR RKDDLHITEE FERLIVTAQM FLPQPDNVRK LSRFYRLDPL DEWRVEVTYK AQKMPAGAFK MTDFHGGKGV ICKV MEDED MPIDENGNRA DLIIFGGSTM RRSNYGRIYE HGFGAAARDL AQRLRVEAGL DRHAKPTQQQ LNSVMGNTQW VDYAF KELL GFYEIIAPTM HSKMMEHPNP AEHVKTVLMD GFPYIYAPVD DPVDLMAAVN KLINSDKYRP HYGKVSYRDQ AGKWVT TKD NVLMGPLYMM LLEKIGEDWS AAASVKTQPF GLPSKLNNAD RASTPGRETA IRSFGESETR SYNCTVGPGP TAEILDQ TN NPLAHAAVIE SWLTAEKPSS VPVAVDREKI PFGGSRPVAM FDHLLECSGI ALEYAPDH

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Macromolecule #4: PHIKZ074

MacromoleculeName: PHIKZ074 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage phiKZ (virus)
Molecular weightTheoretical: 77.513461 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNLNRYKARD LLNLSYDDLW SLPSEWHLIE FDDGKTVVSV DRITKLSVLC WYPLKHYKDC PIPSDHHIDF NRILTDNPKD YLNVEGGRV TSKAMVKHLN KAIWNIYDWS GETVDPEVLS KLAIEGKNWL YNQTTVKLSE YLATLSMFDI AEVYNHPKVR E ANHNIEPT ...String:
MNLNRYKARD LLNLSYDDLW SLPSEWHLIE FDDGKTVVSV DRITKLSVLC WYPLKHYKDC PIPSDHHIDF NRILTDNPKD YLNVEGGRV TSKAMVKHLN KAIWNIYDWS GETVDPEVLS KLAIEGKNWL YNQTTVKLSE YLATLSMFDI AEVYNHPKVR E ANHNIEPT TYGIEKISYG KVKEVFNDPT QFIGNSIIEG LRSGTQKTEQ LLQAFAWRGF PTDINSDIFK YPVTTGYIDG IW NLYENMI ESRSGTKALL YNKELLRVTE YFNRKSQLIA QYVQRLHPGD CKTTILAEYP VTKLTLKAFK GKYYQKEDGK LDW IRGNET HLIGTKQKFR SVFGCNHPDS QGICMTCYGR LGINIPKGTN IGQVAAVSMG DKITSAVLST KHTDASSAVE QYKL GKIES NYLRTGEIPE TLYLKKELTQ KDYRLVIARS EAENLADILM IDDLTAYPAT SATELTSLAL VYDDEVNGEC GDVLT VSLY NRRASLSIEM LKHIKMVRWE LDQRDNIVIS LRGFDFNLPF LTLPNKHVNM YEVMKRFQSF LHSGSDSAEA GKLSTE KVG YTSKTYLKNY KSPIEALPVF ATMANEKISL NISHCEILIY AMMIRSAQYR DYRLPKPGIN GQFEKYNRLM QCRSLGG AM AFEKQHEPLN NPGSFLNKMR NDHPYDLLVK GGKLR

UniProtKB: PHIKZ074

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Macromolecule #5: PHIKZ123

MacromoleculeName: PHIKZ123 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage phiKZ (virus)
Molecular weightTheoretical: 62.95909 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPDPFLIEKI RENTPCMNPT LANGITVEHT MTRDPNTGVN MTRRYIDSLF DISSVLFPDG FKYEGNRACT PLKHFEEITR EYNAKRIAN IAPTDMYMID LMFSYKGEML YPRPMLLPAF KRGNMVTING AKYIGSPVLT DVGFSVLNDS IFIPFRRTKL T FKQTDHHY ...String:
MPDPFLIEKI RENTPCMNPT LANGITVEHT MTRDPNTGVN MTRRYIDSLF DISSVLFPDG FKYEGNRACT PLKHFEEITR EYNAKRIAN IAPTDMYMID LMFSYKGEML YPRPMLLPAF KRGNMVTING AKYIGSPVLT DVGFSVLNDS IFIPFRRTKL T FKQTDHHY MCNGQRKIMY VIWSQIHNEM AKRTKRDLGN RPHIESCLAH YFFCQFGVTQ TFKQWANVDV KCGLLSDFPE EE YPREKWN IYSSATLKGK HPTGEMVLVI PRHQESIFAT RLIAGFWYVV DAFPMRFTRP EYVDSTNLWR VILGHMVFGD FEH QGKVEE NIDSHLHSFC NSLDEMTIEE LKTVGVNVST IWELLYEIMT SLAHHLYATD IDETSMYGKR LTVLHYLMSE FNYA VSMFG YMFQSRRDRE WTVQELNEGL KRSFKLQTAI KRLTVDHGEL DTMSNPNSSM LIKGTSILVT QDRAKTAKAH NKSLI NDSS RIIHASIAEV GQYKNQPKNN PDGRGRLNMY TKVGPTGLVE RREEVREIID NAQLMFRAK

UniProtKB: PHIKZ123

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.3 mg/mL
BufferpH: 8
Details: 15 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 2 mM MgCl2, 1 mM DTT
GridModel: Quantifoil R2/1 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 1
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV / Details: 1-2 s blot.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.25 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 48000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 70 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 100.0 K / Max: 100.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5096 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 10582 / Average electron dose: 40.0 e/Å2 / Details: TIFF movie mode
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3179799
Details: EMAN BATCHBOXER with low-pass butterworth preprocessing
Startup modelType of model: OTHER / Details: RELION - STOCHASTIC GRADIENT DESCENT
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3)
Final 3D classificationNumber classes: 9 / Software - Name: RELION (ver. 3) / Details: 855016 particles classified
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 50085
DetailsMotion correction and dose-weighting in motioncor2
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6edt


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsCore conserved regions initially identified from comparison with PDB 6EDT - remainder built de novo
RefinementProtocol: OTHER
Output model

PDB-7ogp:
Structure of the apo-state of the bacteriophage PhiKZ non-virion RNA polymerase - class including clamp

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