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- EMDB-66346: Cryo-EM structure of the type III-D2 CRISPR-Cas effector complex ... -

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Entry
Database: EMDB / ID: EMD-66346
TitleCryo-EM structure of the type III-D2 CRISPR-Cas effector complex bound to a cognate target RNA in the pre-cleavage state
Map data
Sample
  • Complex: Cryo-EM structure of the type III-D2 CRISPR-Cas effector complex bound to a cognate target RNA in the pre-cleavage state
    • Complex: RNA
      • RNA: CRISPR RNA
      • RNA: Cognate target RNA (CTR)
    • Complex: Cas7x3-Cas7-5-Cas10
      • Protein or peptide: Cas10
      • Protein or peptide: CRISPR type III-associated protein domain-containing protein
      • Protein or peptide: TIGR03986 family CRISPR-associated RAMP protein
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: S-ADENOSYLMETHIONINE
  • Ligand: ZINC ION
  • Ligand: water
KeywordsCRISPR / Anti-phage defense / Second messenger / SAM-AMP / IMMUNE SYSTEM
Function / homology: / : / :
Function and homology information
Biological speciesGammaproteobacteria bacterium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsMitsuda Y / Ishikawa J / Nagahata N / Hiraizumi M / Yamashita K / Nishimasu H
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJCR23B6 Japan
Japan Society for the Promotion of Science (JSPS)25H00436 Japan
CitationJournal: Nat Commun / Year: 2026
Title: Structural mechanism of SAM-AMP and SAM-AMP synthesis by the type III-D2 CRISPR effector complex.
Authors: Yoshihisa Mitsuda / Maki Sugaya / Junichiro Ishikawa / Naoto Nagahata / Sae Okazaki / Masahiro Hiraizumi / Kazuki Kato / Jonathan S Gootenberg / Omar O Abudayyeh / Tsuyoshi Osawa / Keitaro ...Authors: Yoshihisa Mitsuda / Maki Sugaya / Junichiro Ishikawa / Naoto Nagahata / Sae Okazaki / Masahiro Hiraizumi / Kazuki Kato / Jonathan S Gootenberg / Omar O Abudayyeh / Tsuyoshi Osawa / Keitaro Yamashita / Hiroshi Nishimasu /
Abstract: The type III-D2 CRISPR-Cas system comprises multiple Cas subunits and a CRISPR RNA, and is likely an evolutionary intermediate between the well-studied type III-A and III-E systems. Here we show that ...The type III-D2 CRISPR-Cas system comprises multiple Cas subunits and a CRISPR RNA, and is likely an evolutionary intermediate between the well-studied type III-A and III-E systems. Here we show that the type III-D2 complex synthesizes two distinct second messengers, SAM-AMP and SAM-AMP, from S-adenosylmethionine (SAM) and ATP in response to target RNA recognition. We determined cryo-electron microscopy structures of the type III-D2 effector complex in different functional states, providing mechanistic insights into target RNA cleavage and second messenger synthesis. The structures reveal how SAM and ATP are recognized by the Cas10 subunit within the effector complex. Furthermore, our biological data suggest that both SAM-AMP and SAM-AMP act on the CorA ancillary effector, inducing growth arrest of infected bacterial cells and thereby conferring immunity. Thus, our study establishes the type III-D2 system as a unique anti-phage defense mechanism that employs both SAM-AMP and SAM-AMP as second messengers, expanding the repertoire of second messenger strategies in bacterial defense systems and highlighting the remarkable functional diversity of CRISPR-Cas systems.
History
DepositionSep 25, 2025-
Header (metadata) releaseJun 24, 2026-
Map releaseJun 24, 2026-
UpdateJul 8, 2026-
Current statusJul 8, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_66346.png

Downloads & links

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Map

FileDownload / File: emd_66346.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 300 pix.
= 298.8 Å		1 Å/pix.
x 300 pix.
= 298.8 Å		1 Å/pix.
x 300 pix.
= 298.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.996 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.111
Minimum - Maximum-0.84883296 - 1.5313027
Average (Standard dev.)-0.000061022678 (±0.030045362)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_66346_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_66346_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_66346_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of the type III-D2 CRISPR-Cas effector complex ...

EntireName: Cryo-EM structure of the type III-D2 CRISPR-Cas effector complex bound to a cognate target RNA in the pre-cleavage state
Components
  • Complex: Cryo-EM structure of the type III-D2 CRISPR-Cas effector complex bound to a cognate target RNA in the pre-cleavage state
    • Complex: RNA
      • RNA: CRISPR RNA
      • RNA: Cognate target RNA (CTR)
    • Complex: Cas7x3-Cas7-5-Cas10
      • Protein or peptide: Cas10
      • Protein or peptide: CRISPR type III-associated protein domain-containing protein
      • Protein or peptide: TIGR03986 family CRISPR-associated RAMP protein
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: S-ADENOSYLMETHIONINE
  • Ligand: ZINC ION
  • Ligand: water

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Supramolecule #1: Cryo-EM structure of the type III-D2 CRISPR-Cas effector complex ...

SupramoleculeName: Cryo-EM structure of the type III-D2 CRISPR-Cas effector complex bound to a cognate target RNA in the pre-cleavage state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Gammaproteobacteria bacterium (bacteria)

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Supramolecule #2: RNA

SupramoleculeName: RNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #5

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Supramolecule #3: Cas7x3-Cas7-5-Cas10

SupramoleculeName: Cas7x3-Cas7-5-Cas10 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#4

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Macromolecule #1: CRISPR RNA

MacromoleculeName: CRISPR RNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Gammaproteobacteria bacterium (bacteria)
Molecular weightTheoretical: 13.71808 KDa
SequenceString:
AUUGAAACUU GUCUUCGACA UGGGUAAUCC UCAUGUUUGA AUG

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Macromolecule #5: Cognate target RNA (CTR)

MacromoleculeName: Cognate target RNA (CTR) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Gammaproteobacteria bacterium (bacteria)
Molecular weightTheoretical: 15.180135 KDa
SequenceString:
GGCAUUCAAA CAUGAGGAUU ACCCAUGUCG AAGACAACAA AGUUAGG

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Macromolecule #2: Cas10

MacromoleculeName: Cas10 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gammaproteobacteria bacterium (bacteria)
Molecular weightTheoretical: 67.985797 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MNYYLRVEGV NLGSFVNDTN DLATIRGGSL LLLEAMEDIV EKILGKHSSS LSARESFDQL LDKRNVLSA RIKEAKKKQD HALRKKLEQE VRDIKSQIKH LKGKISCRGK GGSSCRETIS KGASWGLFEL DVDESTAAKI K QGLIGAFN ...String:
MGSSHHHHHH SSGLVPRGSH MNYYLRVEGV NLGSFVNDTN DLATIRGGSL LLLEAMEDIV EKILGKHSSS LSARESFDQL LDKRNVLSA RIKEAKKKQD HALRKKLEQE VRDIKSQIKH LKGKISCRGK GGSSCRETIS KGASWGLFEL DVDESTAAKI K QGLIGAFN DDPKYKYATF VVDLYRPEDG EGYRSRRDKV QTLNRYQQLK TLSLAITNQG KDVCTLNKVT PANDERDLPA NS GEKNRDY ISDSVYQRRE YGRGQNVYTG FYKKNTGIDG VKPTKDLEQL SSDAPYPALE GKIAYIYIDG NKFGQKQKEA DSV TEQRSF DQQTREGRKE VLAGILEQIK EDPDWRCGER LRLETLLWGG DEIIWVVPAW RGWWMLKRFY ELAREQIRLV TKDK NGQDE ITRLFHGSSI IFCKHNAPIH RINTLARSLA DGFAKIENFK KRNMIAYQVL ESFDHAGTNL RDYRKNQLKG LSDKL PDFL VEAEQMEAIE DSISKLKQME FPKRKIYQIV QQYRVGMPDE AEKLRKKITG GDYSELTPEQ KEECDKIATE LEHLKS IFG GSNAYWLHLM ELWDYVSPER VDAELQEGE

UniProtKB: UNIPROTKB: A0A9D0P2E0

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Macromolecule #3: CRISPR type III-associated protein domain-containing protein

MacromoleculeName: CRISPR type III-associated protein domain-containing protein
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gammaproteobacteria bacterium (bacteria)
Molecular weightTheoretical: 70.690969 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MQKATLKIGL TLKGPLITQA SDPGDLGLDM VVATHQDGAH YLPGTLIVGK LRQALEELKE VVSLRGGGPM TDWFKPDLDT WLGRPSQTG LPNTKQLYFS DFDLDAESSS KMRSGLVRDR ITIEAETGAV KPRHIVMIEN PFVSGEEYTF TGTLHFFTTE A ATILKHIK ...String:
MQKATLKIGL TLKGPLITQA SDPGDLGLDM VVATHQDGAH YLPGTLIVGK LRQALEELKE VVSLRGGGPM TDWFKPDLDT WLGRPSQTG LPNTKQLYFS DFDLDAESSS KMRSGLVRDR ITIEAETGAV KPRHIVMIEN PFVSGEEYTF TGTLHFFTTE A ATILKHIK TGFNWFSQVG AMKSVGFGQV VGVGFGEPTY ATITPPASAP PAVTTPYVDL VIKPIYPFCI TGRSLSDNLF ES AAIIPGG AIIGSIATTW SHLVGQHDGR IGDDPERKQL KENFSKLRIT HAFPSTADNK RPVVAPLSLV TIDGDQKTYY DVA RLEAPC LINGQPPDFA LDWKDNEDTL KEYPWPFIRM KEWGWASLSA ELRVRTAIDR DYHRSKKSQL FAYEQVVPSG KAWY SRLDL SRIADDYTRS AVLEQLRTLV EQGVYGLGKT KTPFELELLP ADTVTPMVQS SSDPVAGRLW IVTLQTDTLL GSPEP LDET SGECELEIMY SKAWCELSNG NLRLLRYFAR QKQAGGRYLK EVFQKGHSSY RPWLLTEAGS VFVLEADGDD MEEAQR LIK EWLQQGLPLT NEVRKYYRIE SSRVTWEQCP FLAQNGYGEI AVNLEMDGLA TVLPDDSCNI TPIKRTTTGE EG

UniProtKB: UNIPROTKB: A0A9D0P213

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Macromolecule #4: TIGR03986 family CRISPR-associated RAMP protein

MacromoleculeName: TIGR03986 family CRISPR-associated RAMP protein / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gammaproteobacteria bacterium (bacteria)
Molecular weightTheoretical: 138.380078 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SMTTVKEKSW AFTGLKRWKI ITTLETQTPL HIGSGEVAEI EINDSQGDRR QVQANAIIRG KDDKPIIPGS TLKGKLRSHF ESCLDHSKA LERVFGKEYQ SDEEQGRGGL AEFHDAVCSY VAPGNSYYPN WNEARNTYIE ASTTIDCHTG TAADATLHYN E CVPPGTRF ...String:
SMTTVKEKSW AFTGLKRWKI ITTLETQTPL HIGSGEVAEI EINDSQGDRR QVQANAIIRG KDDKPIIPGS TLKGKLRSHF ESCLDHSKA LERVFGKEYQ SDEEQGRGGL AEFHDAVCSY VAPGNSYYPN WNEARNTYIE ASTTIDCHTG TAADATLHYN E CVPPGTRF LVTVTGAMSD KDAALIVAAL QAFGDETNPI HLGAEEANGK GRMGLFGNVE VSCLDHDDII AWISQGSDAR MA TDKFKPL GKEKVNDLAK NITTPTATGG AQRQHFGIEL KFDGPFLVND PSKYSKGDGD QPAVHQPLTD RSNNPILPAR SFR GAIRAQ AERIIRTMGG ACCDTQSPCQ NSGQLCIACQ MFGTTGWKTT LSISDFTYDG EYRPAKTQQF VAIDRFHGGG KDGA LFSIK YFERPVLKGG ISLKLRNQNA DELSWRKGLL ALLFRDLQEG DITFGFGANK GYGGVEEACI TNADVISTAD IEAFR AKCH ANHADSWCSP VSKPTNRDDK SSLPSINPAT GAGHAFHNPY HFIPIKAPDT STWLDKHKLA TPGSPHSHAY YRSCSD DDK PLHHGRITCK LTAETPLFVG SGDAENQLTD SEAKLKEHYQ LNNKLAIPAT SLRGLISSLA EAASNSALRV LDNGVLS YR KPASRALRKI GILFKREEQW RLVQMEGNLA NAIKLKSAYT NQKMMDFLAN KQSWSPEHNV VYYLSADFRP GDVPQETY L AGRICGILRI LGGKDGDRKN ELENKKHELF IRVDEQYVDT EINRFDYEEY VRQGGIPVSP SAVERYTELA DQRSLSQKN SRDLKGDNNC CSDKWLPFHL KGAARYKKEK ACLLPLREYD LVYFDSDGTQ VTEISFSAIW RDRVADKVHA FFPEELRPFN QKRKWISPA ELLFGFVELN DNKDERDHAQ AFTGKVRVAA GVLSPDDSIR QGDLQEHEPI MLKALSSPKL PSPALYFKQK S GDHRYIAK PDLKKASHQA QGRKIYLHAL RDQKDDVQKL NTKGQPANGN GAHLPWKTAD EDERPQLKVR IRPLKPGTSF YF HLDYNNL TEWELGLLCY VLQPSETFRH KLGMGKPIGL GTVKIEIATL QTIDRQKRYR EAGANEHRHN GSNWVNESLR DEL ERLPGT VELSPDRQPE AKLRPDELRQ SFIATMDNDI YRAIELLGDP HNIKYPVHYP QVRNKSIEQE NFKWFVANDS GSGD QRKGT GIDAKEEPMR SIDQISTTIP TLNRYEWNGD

UniProtKB: UNIPROTKB: A0A9D0P2B2

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #8: S-ADENOSYLMETHIONINE

MacromoleculeName: S-ADENOSYLMETHIONINE / type: ligand / ID: 8 / Number of copies: 1 / Formula: SAM
Molecular weightTheoretical: 398.437 Da
Chemical component information

ChemComp-SAM:
S-ADENOSYLMETHIONINE

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Macromolecule #9: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #10: water

MacromoleculeName: water / type: ligand / ID: 10 / Number of copies: 17 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.15 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMC8H17N2O4SNaHEPES-NaOH
5.0 mMMgCl2magnesium chloride
1.0 mMC15H22N6O5SS-adenosylmethinonine
1.0 mMC10H17N6O12P3Adenylyl imidodiphosphate
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 49.833 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 371927
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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