EMDB-75681: Cryo-EM structure of the bacteriophage N4 virion RNA polymerase (...

Basic information

Entry

Database: EMDB / ID: EMD-75681

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

Sample

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

• Keywords: Bacteriophage N4 phage vRNAP protein / 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: 3.43 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 23, 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_75681.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Full map
• Voxel size: X=Y=Z: 1.07 Å

Density

Contour Level	By AUTHOR: 0.102
Minimum - Maximum	-0.24639311 - 0.5657524
Average (Standard dev.)	0.0017087456 (±0.023214469)

• 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 A

• File: emd_75681_half_map_1.map
• Annotation: Half map A

Half map: Half map B

• File: emd_75681_half_map_2.map
• Annotation: Half map B

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 (closed plug state)

Components

• Complex: Cryo-EM structure of the bacteriophage N4 virion RNA polymerase (closed plug state)
  • 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 (closed plug state); type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
• Source (natural): Organism: Escherichia phage N4 (virus)
• Molecular weight: Theoretical: 274.2 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: 382.9195 KDa
• Recombinant expression: Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

Sequence

String:

MSVFDRLAGF ADSVTNAKQV DVSTATAQKK AEQGVTTPLV SPDAAYQMQA ARTGNVGANA FEPGTVQSDF MNLTPMQIMN KYGVEQGLQ LINARADAGN QVFNDSVTTR TPGEELGDIA TGVGLGFVNT LGGIGALGAG LLNDDAGAVV AQQLSKFNDA V HATQSQAL QDKRKLFAAR NLMNEVESER QYQTDKKEGT NDIVASLSKF GRDFVGSIEN AAQTDSIISD GLAEGVGSLL GA GPVLRGA SLLGKAVVPA NTLRSAALAG AIDAGTGTQS LARIASTVGR AAPGMVGVGA MEAGGAYQQT ADEIMKMSLK DLE KSPVYQ QHIKDGMSPE QARRQTASET GLTAAAIQLP IAAATGPLVS RFEMAPFRAG SLGAVGMNLA RETVEEGVQG ATGQ LAQNI AQQQNIDKNQ DLLKGVGTQA GLGALYGFGS AGVVQAPAGA ARLAGAATAP VLRTTMAGVK AAGSVAGKVV SPIKN TLVA RGERVMKQNE EASPVADDYV AQAAQEAMAQ APEAEVTIRD AVEATDATPE QKVAAHQYVS DLMNATRFNP ENYQEA PEH IRNAVAGSTD QVQVIQKLAD LVNTLDESNP QALMEAASYM YDAVSEFEQF INRDPAALDS IPKDSPAIEL LNRYTNL TA NIQNTPKVIG ALNVINRMIN ESAQNGSLNV TEESSPQEMQ NVALAAEVAP EKLNPESVNV VLKHAADGRI KLNNRQIA A LQNAAAILKG AREYDAEAAR LGLRPQDIVS KQIKTDESRT QEGQYSALQH ANRIRSAYNS GNFELASAYL NDFMQFAQH MQNKVGALNE HLVTGNADKN KSVHYQALTA DREWVRSRTG LGVNPYDTKS VKFAQQVALE AKTVADIANA LASAYPELKV SHIKVTPLD SRLNAPAAEV VKAFRQGNRD VASSQPKADS VNQVKETPVT KQEPVTSTVQ TKTPVSESVK TEPTTKESSP Q AIKEPVNQ SEKQDVNLTN EDNIKQPTES VKETETSTKE STVTEELKEG IDAVYPSLVG TADSKAEGIK NYFKLSFTLP EE QKSRTVG SEAPLKDVAQ ALSSRARYEL FTEKETANPA FNGEVIKRYK ELMEHGEGIA DILRSRLAKF LNTKDVGKRF AQG TEANRW VGGKLLNIVE QDGDTFKYNE QLLQTAVLAG LQWRLTATSN TAIKDAKDVA AITGIDQALL PEGLVEQFDT GMTL TEAVS SLAQKIESYW GLSRNPNAPL GYTKGIPTAM AAEILAAFVE STDVVENIVD MSEIDPDNKK TIGLYTITEL DSFDP INSF PTAIEEAVLV NPTEKMFFGD DIPPVANTQL RNPAVRNTPE QKAALKAEQA TEFYVHTPMV QFYETLGKDR ILELMG AGT LNKELLNDNH AKSLEGKNRS VEDSYNQLFS VIEQVRAQSE DISTVPIHYA YNMTRVGRMQ MLGKYNPQSA KLVREAI LP TKATLDLSNQ NNEDFSAFQL GLAQALDIKV HTMTREVMSD ELTKLLEGNL KPAIDMMVEF NTTGSLPENA VDVLNTAL G DRKSFVALMA LMEYSRYLVA EDKSAFVTPL YVEADGVTNG PINAMMLMTG GLFTPDWIRN IAKGGLFIGS PNKTMNEHR STADNNDLYQ ASTNALMESL GKLRSNYASN MPIQSQIDSL LSLMDLFLPD INLGENGALE LKRGIAKNPL TITIYGSGAR GIAGKLVSS VTDAIYERMS DVLKARAKDP NISAAMAMFG KQAASEAHAE ELLARFLKDM ETLTSTVPVK RKGVLELQST G TGAKGKIN PKTYTIKGEQ LKALQENMLH FFVEPLRNGI TQTVGESLVY STEQLQKATQ IQSVVLEDMF KQRVQEKLAE KA KDPTWKK GDFLTQKELN DIQASLNNLA PMIETGSQTF YIAGSENAEV ANQVLATNLD DRMRVPMSIY APAQAGVAGI PFM TIGTGD GMMMQTLSTM KGAPKNTLKI FDGMNIGLND ITDASRKANE AVYTSWQGNP IKNVYESYAK FMKNVDFSKL SPEA LEAIG KSALEYDQRE NATVDDIANA ASLIERNLRN IALGVDIRHK VLDKVNLSID QMAAVGAPYQ NNGKIDLSNM TPEQQ ADEL NKLFREELEA RKQKVAKARA EVKEETVSEK EPVNPDFGMV GREHKASGVR ILSATAIRNL AKISNLPSTQ AATLAE IQK SLAAKDYKII YGTPTQVAEY ARQKNVTELT SQEMEEAQAG NIYGWTNFDD KTIYLVSPSM ETLIHELVHA STFEEVY SF YQGNEVSPTS KQAIENLEGL MEQFRSLDIS KDSPEMREAY ADAIATIEGH LSNGFVDPAI SKAAALNEFM AWGLANRA L AAKQKRTSSL VQMVKDVYQA IKKLIWGRKQ APALGEDMFS NLLFNSAILM RSQPTTQAVA KDGTLFHSKA YGNNERLSQ LNQTFDKLVT DYLRTDPVTE VERRGNVANA LMSATRLVRD VQSHGFNMTA QEQSVFQMVT AALATEAAID PHAMARAQEL YTHVMKHLT VEHFMADPDS TNPADRYYAQ QKYDTISGAN LVEVDAKGRT SLLPTFLGLA MVNEELRSII KEMPVPKADK K LGNDIDTL LTNAGTQVME SLNRRMAGDQ KATNVQDSID ALSETIMAAA LKRESFYDAV ATPTGNFIDR ANQYVTDSIE RL SETVIEK ADKVIANPSN IAAKGVAHLA KLTAAIASEK QGEIVAQGVM TAMNQGKVWQ PFHDLVNDIV GRTKTNANVY DLI KLVKSQ ISQDRQQFRE HLPTVIAGKF SRKLTDTEWS AMHTGLGKTD LAVLRETMSM AEIRDLLSSS KKVKDEISTL EKEI QNQAG RNWNLVQKKS KQLAQYMIMG EVGNNLLRNA HAISRLLGER ITNGPVADVA AIDKLITLYS LELMNKSDRD LLSEL AQSE VEGMEFSIAY MVGQRTEEMR KAKGDNRTLL NHFKGYIPVE NQQGVNLIIA DDKEFAKLNS QSFTRIGTYQ GSTGFR TGS KGYYFSPVAA RAPYSQGILQ NVRNTAGGVD IGTGFTLGTM VAGRITDKPT VERITKALAK GERGREPLMP IYNSKGQ VV AYEQSVDPNM LKHLNQDNHF AKMVGVWRGR QVEEAKAQRF NDILIEQLHA MYEKDIKDSS ANKSQYVNLL GKIDDPVL A DAINLMNIET RHKAEELFGK DELWVRRDML NDALGYRAAS IGDVWTGNSR WSPSTLDTVK KMFLGAFGNK AYHVVMNAE NTIQNLVKDA KTVIVVKSVV VPAVNFLANI YQMIGRGVPV KDIAVNIPRK TSEINQYIKS RLRQIDAEAE LRAAEGNPNL VRKLKTEIQ SITDSHRRMS IWPLIEAGEF SSIADAGISR DDLLVAEGKI HEYMEKLANK LPEKVRNAGR YALIAKDTAL F QGIQKTVE YSDFIAKAII YDDLVKRKKK SSSEALGQVT EEFINYDRLP GRFRGYMESM GLMWFYNFKI RSIKVAMSMI RN NPVHSLI ATVVPAPTMF GNVGLPIQDN MLTMLAEGRL DYSLGFGQGL RAPTLNPWFN LTH

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 R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
• Vitrification: Cryogen name: ETHANE

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 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
• Startup model: Type of model: INSILICO MODEL
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 50000
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC