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- EMDB-66260: Cryo-EM structure of Upx -

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Basic information

Entry
Database: EMDB / ID: EMD-66260
TitleCryo-EM structure of Upx
Map data
Sample
  • Cell: protein structure
    • Protein or peptide: Restriction endonuclease
Keywordsprotein structure / ANTIVIRAL PROTEIN
Function / homology: / : / Anti-phage protein Upx-like / Uncharacterized protein
Function and homology information
Biological speciesEscherichia coli (E. coli) / Salmonella enterica subsp. enterica serovar Bredeney (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsZhang H / Li X
Funding support1 items
OrganizationGrant numberCountry
Other government
CitationJournal: Nat Commun / Year: 2026
Title: Structural and mechanistic insights into the dual-nuclease defense protein Upx as an anti-phage system.
Authors: Ruimin Zhou / Yingcan Liu / Qian Zhang / Zhenning Yin / Jiajing Tong / Chendi Zhang / Lingling Zhang / Xuzichao Li / Yanhao Zhao / Shuqin Zhang / Zhikun Liu / Weichang Chen / Nan Ji / Heng ...Authors: Ruimin Zhou / Yingcan Liu / Qian Zhang / Zhenning Yin / Jiajing Tong / Chendi Zhang / Lingling Zhang / Xuzichao Li / Yanhao Zhao / Shuqin Zhang / Zhikun Liu / Weichang Chen / Nan Ji / Heng Zhang / Zhuang Li / Hang Yin / Shengkai Zuo / Yong Wei /
Abstract: Nucleic acid degradation is a common strategy for prokaryotic anti-phage systems, as exemplified by the CRISPR-Cas system. The PD-(D/E)-XK nucleases constitute a widely distributed family in these ...Nucleic acid degradation is a common strategy for prokaryotic anti-phage systems, as exemplified by the CRISPR-Cas system. The PD-(D/E)-XK nucleases constitute a widely distributed family in these defenses. Notably, most members exhibit a single nuclease domain, while variants containing dual nuclease domains within a single polypeptide remain underexplored, and their molecular mechanisms largely obscure. Here, we biochemically and functionally study a single-protein system containing an uncharacterized PD-(D/E)-XK defense protein (Upx). As revealed by single-particle electron cryo-microscopy (cryo-EM) structure, the C-terminal domain (CTD) harboring the conserved PD-(D/E)XK catalytic core is buttressed by the N-terminal domain (NTD) and the middle domain (MD). Functional assays demonstrate that the nucleic acid binding capability of the CTD is enhanced by the MD. The NTD also displays a noncanonical, basal exonuclease activity that is auto-inhibited by MD. IP-MS experiments identify Upx-interacting phage proteins, and substrate profiling defines its physiological preferences, collectively pointing to its potential physiological targets. Notably, the phage protein gp16 was found to relieve MD-mediated inhibition of the NTD, suggesting a virus-triggered mechanism for activating Upx's dual nuclease activity. Together, these findings establish Upx as a single-protein dual-nuclease anti-phage system, expanding our understanding of bacterial immunity and informing antiviral strategy development.
History
DepositionSep 18, 2025-
Header (metadata) releaseFeb 11, 2026-
Map releaseFeb 11, 2026-
UpdateAug 26, 2026-
Current statusAug 26, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_66260.png

Downloads & links

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Map

FileDownload / File: emd_66260.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.69 Å/pix.
x 350 pix.
= 242.2 Å		0.69 Å/pix.
x 350 pix.
= 242.2 Å		0.69 Å/pix.
x 350 pix.
= 242.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.692 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.27196375 - 0.7088378
Average (Standard dev.)0.00077457144 (±0.021281183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 242.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_66260_half_map_1.map
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Half map: #2

Fileemd_66260_half_map_2.map
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Sample components

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Entire : protein structure

EntireName: protein structure
Components
  • Cell: protein structure
    • Protein or peptide: Restriction endonuclease

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Supramolecule #1: protein structure

SupramoleculeName: protein structure / type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Restriction endonuclease

MacromoleculeName: Restriction endonuclease / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Bredeney (bacteria)
Molecular weightTheoretical: 143.0245 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNEHLSHMDV HTLFEEMDEQ ADGITFKYSF DDIAKSNALV VTEFVNFERD STVALLASLL TLPAHQSQCL RFELLTSLAL IHCKGQQIA NIDDVKRWYV TIGESSSIVG EDPAEDVFVA LVDNKKGDYR VLEGVWEAAG FYTQLMVEIV SDMPDTHRYR S LKLAIQAI ...String:
MNEHLSHMDV HTLFEEMDEQ ADGITFKYSF DDIAKSNALV VTEFVNFERD STVALLASLL TLPAHQSQCL RFELLTSLAL IHCKGQQIA NIDDVKRWYV TIGESSSIVG EDPAEDVFVA LVDNKKGDYR VLEGVWEAAG FYTQLMVEIV SDMPDTHRYR S LKLAIQAI LRLSDVICAR SGLYRFQEGA DEFPDSLDTA GLDEKTLCSR VTLSERSLRA EGIKLADLAP FILEPSHISM LG NQVPGEG MLEQRPLLRT RDGIVVVLPT AMTIALRQAV ITFAKRTEEL SELDKALANV YSLTFSEMPV FGNGGRLRRL TWE KYKMSR TTMVTSIVDA GHLMVLQFVL PSIQQYADTG FNNLLQLDEE TTQFLDNSVE QITVDLAKQP GFQRGIVVRI ACGW GAGFM GVPPQLPDGW GFEWMSGADF VRFGALPDMS PIAFWRVQDA VETIRQAGVR LINMSGTLNL LGWIRANDGH MVPHD QLPD DRITPEHPLM LMIPTNLLRG IRIAADTGYD RHRISDNNGK WHRVMRPSAE DFFPTERQSK CYASIDDLEA QRLTCV YEG QGNLWVTLEA PEMEDWMLLV ELAKMVRTWI GRIGEALEVL SEQPIKKSLK VYLHFDGNDN IGRFDGENFS DDMNTFW RL ERIHEHGAIR VVLQDGYLAG FRLPDNRAER ALVRALGTAF ATLLRMKEPV DKGVTVEQIA VPNDRARSFH IMQAYDFN Q YLGRSLTKRL LAIEDIDSAA ARIELAWRAV STDAPSRYQG KKEVGKLLND VVDVLIQDLL SELSRFDRKQ TVMRLLENV VKARCEEAHW RSTAAAVLGL HAGEEGVEET IAQEMSRYAG AALTSRLIIE LAICVCPTSG GIEPSDMALS KLLARASLLF RIGGMSDAV RFGALPADIR ISPLGDLLFR DELGKMVLEP MLSKVTNERF EEQAAQFEQH YVKTAGGDDE NSKQDSVAAE T TEDQTDIF LAFWKAEMGF TLEDGMRFIQ FLESIGIEQE SAIFEMRRSQ LADAAKSAGL ADETIDAFLN QFILSARPKW DV VPDGFDL SDIYPWRFGR RLSVAVRPLL QIEESHDPLI VIAPGLLNLS LKYVFDGAYT GQFKRDFFRT EGMRDTWLGG ARE GHTFEK TLERELREIG WTVRRGIGFP EILRRNLPGD PGDIDLLAWR SDRNQVLVIE CKDLSLARNY SEVASQLSEY QGDD IKGKP DKLKKHLKRV LLAKENIDNF AKFTSIANPE IVSWLVFSGA SPIAYAQSKI EALAGTNVGR PSDLLNF

UniProtKB: Uncharacterized protein

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 204860
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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