EMDB-75669: Cryo-EM structure of the bacteriophage N4 virion RNA polymerase (...

Basic information

Entry

Database: EMDB / ID: EMD-75669

• Title: Cryo-EM structure of the bacteriophage N4 virion RNA polymerase (open plug state)
• Map data: Full EM map

Sample

• Complex: Cryo-EM structure of the bacteriophage N4 virion RNA polymerase
  • Protein or peptide: Virion DNA-directed RNA polymerase

• Keywords: N4 phage viral RNA polymerase / VIRAL PROTEIN

Function / homology

Function:

DNA-directed RNA polymerase complex / virion component / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / GTP binding / ATP binding / metal ion binding

Domain/homology:

: / : / : / : / Virion DNA-directed RNA polymerase, plug insertion / Virion DNA-directed RNA polymerase domain / Virion DNA-directed RNA polymerase domain / Bacteriophage N4 RNA polymerase, helical domain

Component:

Virion DNA-directed RNA polymerase

• Biological species: Escherichia phage N4 (virus)
• Method: single particle reconstruction / cryo EM / Resolution: 3.35 Å
• Authors: Narwal M / Shin Y / Murakami KS

Funding support

1 items

Organization	Grant number	Country
Not funded

• Citation: Journal: J Bacteriol / Year: 2026; Title: Cryo-EM study of bacteriophage N4 virion RNA polymerase.; Authors: Manju Narwal / Yeonoh Shin / Katsuhiko S Murakami / ; Abstract: Coliphage N4 employs a unique infection and transcription strategy in which early gene expression is driven by a virion-encapsidated RNA polymerase (vRNAP) that is injected into the host cytoplasm upon infection. Despite extensive biochemical and crystallographic studies of the polymerase domain of vRNAP, the structural organization and regulatory roles of the N-terminal domain (NTD) and C-terminal domain (CTD) regions of the 3,500-residue-long whole enzyme have remained unresolved. Here, we report the cryo-electron microscopy (cryo-EM) structures of full-length N4 vRNAP in its apo state and in a transcription initiation complex (TIC) with promoter DNA and initiating nucleotides. The apo structure reveals a modular architecture in which an α-helical CTD packs against the Pol domain to stabilize an autoinhibited conformation characterized by occlusion of the nucleotide-binding site through tight contact between the plug module and motif B loop. In contrast, promoter binding induces conformational rearrangements that displace the motif B loop from the active site and separate the CTD from the Pol domain. The NTD is unresolved in both states, consistent with substantial intrinsic flexibility, and supporting its proposed role in membrane association and genome injection. Structural modeling suggests that domain segmentation and conformational plasticity may enable translocation of vRNAP through the ~30 Å wide phage tail channel during infection. Together, these results define the molecular architecture of full-length vRNAP and establish a structural framework for understanding how the conformational transition of vRNAP is coupled to its ejection, DNA injection, and early gene expression.IMPORTANCEThis study investigates the structure of full-length bacteriophage N4 virion RNA polymerase (vRNAP), one of the largest known single-subunit RNA polymerases. The functions of its extensive N- and C-terminal regions remained unknown. Our work uncovers how the C-terminal domain regulates polymerase activity through a structural "switch" that locks the enzyme in an inactive state until it recognizes its promoter DNA. These findings explain how the phage prevents premature transcription and ensures precise control of early gene expression during infection. By integrating structures with the architecture of the N4 phage particle, we propose a mechanism by which this vRNAP is transported through the narrow phage tail into the host cell. Together, this work provides fundamental insight into phage transcription and viral gene regulation.

History

Deposition	Feb 21, 2026	-
Header (metadata) release	Jun 24, 2026	-
Map release	Jun 24, 2026	-
Update	Aug 12, 2026	-
Current status	Aug 12, 2026	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_75669.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Full EM map
• Voxel size: X=Y=Z: 1.07 Å

Density

Contour Level	By AUTHOR: 0.102
Minimum - Maximum	-0.2754007 - 0.5975636
Average (Standard dev.)	0.001648687 (±0.024028452)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 240 240 240 Spacing 240 240 240
Cell	A=B=C: 256.80002 Å α=β=γ: 90.0 °

Supplemental data

Half map: Half map B

• File: emd_75669_half_map_1.map
• Annotation: Half map B

Half map: Half map A

• File: emd_75669_half_map_2.map
• Annotation: Half map A

Sample components

Entire : Cryo-EM structure of the bacteriophage N4 virion RNA polymerase

• Entire: Name: Cryo-EM structure of the bacteriophage N4 virion RNA polymerase

Components

• Complex: Cryo-EM structure of the bacteriophage N4 virion RNA polymerase
  • Protein or peptide: Virion DNA-directed RNA polymerase

Supramolecule #1: Cryo-EM structure of the bacteriophage N4 virion RNA polymerase

• Supramolecule: Name: Cryo-EM structure of the bacteriophage N4 virion RNA polymerase; type: complex / ID: 1 / Parent: 0 / Macromolecule list: all; Details: Cryo-EM structure of the bacteriophage N4 virion RNA polymerase in an open plug state
• Source (natural): Organism: Escherichia phage N4 (virus)
• Molecular weight: Theoretical: 380 KDa

Macromolecule #1: Virion DNA-directed RNA polymerase

• Macromolecule: Name: Virion DNA-directed RNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
• Source (natural): Organism: Escherichia phage N4 (virus)
• Molecular weight: Theoretical: 276.245375 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

GIDAVYPSLV GTADSKAEGI KNYFKLSFTL PEEQKSRTVG SEAPLKDVAQ ALSSRARYEL FTEKETANPA FNGEVIKRYK ELMEHGEGI ADILRSRLAK FLNTKDVGKR FAQGTEANRW VGGKLLNIVE QDGDTFKYNE QLLQTAVLAG LQWRLTATSN T AIKDAKDV AAITGIDQAL LPEGLVEQFD TGMTLTEAVS SLAQKIESYW GLSRNPNAPL GYTKGIPTAM AAEILAAFVE ST DVVENIV DMSEIDPDNK KTIGLYTITE LDSFDPINSF PTAIEEAVLV NPTEKMFFGD DIPPVANTQL RNPAVRNTPE QKA ALKAEQ ATEFYVHTPM VQFYETLGKD RILELMGAGT LNKELLNDNH AKSLEGKNRS VEDSYNQLFS VIEQVRAQSE DIST VPIHY AYNMTRVGRM QMLGKYNPQS AKLVREAILP TKATLDLSNQ NNEDFSAFQL GLAQALDIKV HTMTREVMSD ELTKL LEGN LKPAIDMMVE FNTTGSLPEN AVDVLNTALG DRKSFVALMA LMEYSRYLVA EDKSAFVTPL YVEADGVTNG PINAMM LMT GGLFTPDWIR NIAKGGLFIG SPNKTMNEHR STADNNDLYQ ASTNALMESL GKLRSNYASN MPIQSQIDSL LSLMDLF LP DINLGENGAL ELKRGIAKNP LTITIYGSGA RGIAGKLVSS VTDAIYERMS DVLKARAKDP NISAAMAMFG KQAASEAH A EELLARFLKD METLTSTVPV KRKGVLELQS TGTGAKGKIN PKTYTIKGEQ LKALQENMLH FFVEPLRNGI TQTVGESLV YSTEQLQKAT QIQSVVLEDM FKQRVQEKLA EKAKDPTWKK GDFLTQKELN DIQASLNNLA PMIETGSQTF YIAGSENAEV ANQVLATNL DDRMRVPMSI YAPAQAGVAG IPFMTIGTGD GMMMQTLSTM KGAPKNTLKI FDGMNIGLND ITDASRKANE A VYTSWQGN PIKNVYESYA KFMKNVDFSK LSPEALEAIG KSALEYDQRE NATVDDIANA ASLIERNLRN IALGVDIRHK VL DKVNLSI DQMAAVGAPY QNNGKIDLSN MTPEQQADEL NKLFREELEA RKQKVAKARA EVKEETVSEK EPVNPDFGMV GRE HKASGV RILSATAIRN LAKISNLPST QAATLAEIQK SLAAKDYKII YGTPTQVAEY ARQKNVTELT SQEMEEAQAG NIYG WTNFD DKTIYLVSPS METLIHELVH ASTFEEVYSF YQGNEVSPTS KQAIENLEGL MEQFRSLDIS KDSPEMREAY ADAIA TIEG HLSNGFVDPA ISKAAALNEF MAWGLANRAL AAKQKRTSSL VQMVKDVYQA IKKLIWGRKQ APALGEDMFS NLLFNS AIL MRSQPTTQAV AKDGTLFHSK AYGNNERLSQ LNQTFDKLVT DYLRTDPVTE VERRGNVANA LMSATRLVRD VQSHGFN MT AQEQSVFQMV TAALATEAAI DPHAMARAQE LYTHVMKHLT VEHFMADPDS TNPADRYYAQ QKYDTISGAN LVEVDAKG R TSLLPTFLGL AMVNEELRSI IKEMPVPKAD KKLGNDIDTL LTNAGTQVME SLNRRMAGDQ KATNVQDSID ALSETIMAA ALKRESFYDA VATPTGNFID RANQYVTDSI ERLSETVIEK ADKVIANPSN IAAKGVAHLA KLTAAIASEK QGEIVAQGVM TAMNQGKVW QPFHDLVNDI VGRTKTNANV YDLIKLVKSQ ISQDRQQFRE HLPTVIAGKF SRKLTDTEWS AMHTGLGKTD L AVLRETMS MAEIRDLLSS SKKVKDEIST LEKEIQNQAG RNWNLVQKKS KQLAQYMIMG EVGNNLLRNA HAISRLLGER IT NGPVADV AAIDKLITLY SLELMNKSDR DLLSELAQSE VEGMEFSIAY MVGQRTEEMR KAKGDNRTLL NHFKGYIPVE NQQ GVNLII ADDKEFAKLN SQSFTRIGTY QGSTGFRTGS KGYYFSPVAA RAPYSQGILQ NVRNTAGGVD IGTGFTLGTM VAGR ITDKP TVERITKALA KGERGREPLM PIYNSKGQVV AYEQSVDPNM LKHLNQDNHF AKMVGVWRGR QVEEAKAQRF NDILI EQLH AMYEKDIKDS SANKSQYVNL LGKIDDPVLA DAINLMNIET RHKAEELFGK DELWVRRDML NDALGYRAAS IGDVWT GNS RWSPSTLDTV KKMFLGAFGN KAYHVVMNAE NTIQNLVKDA KTVIVVKSVV VPAVNFLANI YQMIGRGVPV KDIAVNI PR KTSEINQYIK SRLRQIDAEA ELRAAEGNPN LVRKLKTEIQ SITDSHRRMS IWPLIEAGEF SSIADAGISR DDLLVAEG K IHEYMEKLAN KLPEKVRNAG RYALIAKDTA LFQGIQKTVE YSDFIAKAII YDDLVKRKKK SSSEALGQVT EEFINYDRL PGRFRGYMES MGLMWFYNFK IRSIKVAMSM IRNNPVHSLI ATVVPAPTMF GNVGLPIQDN MLTMLAEGRL DYSLGFGQGL RAPTLNPWF NLTH

UniProtKB: Virion DNA-directed RNA polymerase

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 1.5 mg/mL
• Buffer: pH: 8
• Grid: Model: Quantifoil Active R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
• Details: Monodisperse sample of the vRNAP full length protein with all the domains intact

Electron microscopy

• Microscope: TFS KRIOS
• Image recording: Film or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• CTF correction: Software - Name: cryoSPARC (ver. 4.71) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
• Startup model: Type of model: NONE
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.71) / Number images used: 57669
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.71)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.71)
• Final 3D classification: Software - Name: cryoSPARC (ver. 4.71)