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- EMDB-66472: Target analog-bound type III-B Cmr complex of Archaeoglobus fulgidus -

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Basic information

Entry
Database: EMDB / ID: EMD-66472
TitleTarget analog-bound type III-B Cmr complex of Archaeoglobus fulgidus
Map dataPrimary map
Sample
  • Complex: Archaeoglobus fulgidus type III-B Cmr CRISPR-Cas effector
    • RNA: crRNA
    • Protein or peptide: GGDEF domain-containing protein
    • Protein or peptide: Type III-B CRISPR module-associated protein Cmr3
    • Protein or peptide: CRISPR type III-associated protein domain-containing protein
    • Protein or peptide: CRISPR system Cmr subunit Cmr5
    • Protein or peptide: CRISPR type III-associated protein domain-containing protein
    • DNA: Target analog DNA
KeywordsType III-B CRISPR-Cas effector / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


defense response to virus / cytoplasm
Similarity search - Function
CRISPR-associated protein, TM1793 / CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, TM1791 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 / AF1862-like domain superfamily / CRISPR-associated protein Cmr2 / CRISPR-associated protein Cmr2, N-terminal ...CRISPR-associated protein, TM1793 / CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, TM1791 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 / AF1862-like domain superfamily / CRISPR-associated protein Cmr2 / CRISPR-associated protein Cmr2, N-terminal / CRISPR-Cas system, Cmr2 subunit, D1 domain, cysteine cluster / CRISPR-associated protein Cmr2, N-terminal / : / : / Cas10/Cmr2, second palm domain / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
GGDEF domain-containing protein / Type III-B CRISPR module-associated protein Cmr3 / CRISPR type III-associated protein domain-containing protein / CRISPR system Cmr subunit Cmr5 / CRISPR type III-associated protein domain-containing protein
Similarity search - Component
Biological speciesArchaeoglobus fulgidus DSM 4304 (archaea) / Pyrococcus furiosus DSM 3638 (archaea) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsIshihara K / Numata T
Funding support Japan, 3 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H02916 Japan
Japan Society for the Promotion of Science (JSPS)24H00505 Japan
Japan Society for the Promotion of Science (JSPS)23KJ1734 Japan
CitationJournal: Biochem Biophys Res Commun / Year: 2025
Title: Cryo-EM structure of Archaeoglobus fulgidus type III-B CRISPR-Cas effector and intermediate crRNA processing during effector assembly.
Authors: Kazuki Ishihara / Sumire Kitagawa / Naruhiko Adachi / Masato Akutsu / Toshiya Senda / Hideko Inanaga / Tomoyuki Numata /
Abstract: Type III CRISPR-Cas effectors recognize target RNAs complementary to the crRNA guide, activating diverse downstream antiviral responses. In contrast to type III-A systems, the architecture of the ...Type III CRISPR-Cas effectors recognize target RNAs complementary to the crRNA guide, activating diverse downstream antiviral responses. In contrast to type III-A systems, the architecture of the type III-B effector (Cmr), comprising six proteins (Cmr1-Cmr6) and a crRNA, remains incompletely defined. Moreover, although maturation of the 3' region of type III crRNA has been attributed to polynucleotide phosphorylase (PNPase), an alternative maturation pathway has been suggested but remains to be elucidated. Here we determined the cryo-EM structure of the Cmr1-lacking Archaeoglobus fulgidus Cmr (AfCmrΔ1) bound to a target analog at 3.4 Å resolution. The complex forms a continuous basic channel that accommodates a crRNA-target heteroduplex. Comparative interface analysis explains why the previously reported cross-species Cmr assembly retains activity, revealing interface flexibility that enables compatible Cmr3-Cmr4 and Cmr2-Cmr5 interactions. Furthermore, we show the cooperative, site-specific processing of an intermediate crRNA that requires both AfCmrΔ1 and AfCmr1 and proceeds without divalent cations. In addition to identifying the cleavage site within the intermediate crRNA, mutational analysis of AfCmr1 reveals residues critical for the reaction. These findings suggest an alternative pathway for crRNA maturation during type III effector assembly that complements PNPase-mediated trimming of the intermediate crRNA, thereby expanding the mechanistic landscape of type III CRISPR-Cas systems.
History
DepositionOct 3, 2025-
Header (metadata) releaseDec 10, 2025-
Map releaseDec 10, 2025-
UpdateDec 10, 2025-
Current statusDec 10, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_66472.png

Downloads & links

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Map

FileDownload / File: emd_66472.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 500 pix.
= 440. Å		0.88 Å/pix.
x 500 pix.
= 440. Å		0.88 Å/pix.
x 500 pix.
= 440. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.88 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.27
Minimum - Maximum-0.21114913 - 0.96737355
Average (Standard dev.)-0.00010445946 (±0.025477823)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 440.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_66472_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

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Additional map: Sharpened map

Fileemd_66472_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_66472_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_66472_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : Archaeoglobus fulgidus type III-B Cmr CRISPR-Cas effector

EntireName: Archaeoglobus fulgidus type III-B Cmr CRISPR-Cas effector
Components
  • Complex: Archaeoglobus fulgidus type III-B Cmr CRISPR-Cas effector
    • RNA: crRNA
    • Protein or peptide: GGDEF domain-containing protein
    • Protein or peptide: Type III-B CRISPR module-associated protein Cmr3
    • Protein or peptide: CRISPR type III-associated protein domain-containing protein
    • Protein or peptide: CRISPR system Cmr subunit Cmr5
    • Protein or peptide: CRISPR type III-associated protein domain-containing protein
    • DNA: Target analog DNA

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Supramolecule #1: Archaeoglobus fulgidus type III-B Cmr CRISPR-Cas effector

SupramoleculeName: Archaeoglobus fulgidus type III-B Cmr CRISPR-Cas effector
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Archaeoglobus fulgidus DSM 4304 (archaea)
Molecular weightTheoretical: 370 KDa

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Macromolecule #1: crRNA

MacromoleculeName: crRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 12.604483 KDa
SequenceString:
AUUGAAAGUU GUAGUAUGCG GUCCUUGCGG CUGAGAGCA

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Macromolecule #2: GGDEF domain-containing protein

MacromoleculeName: GGDEF domain-containing protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Archaeoglobus fulgidus DSM 4304 (archaea)
Molecular weightTheoretical: 117.383156 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MGQYFRVSGK YRGRENLPRE VNKMADNEFW LNKIRAFFHD PPDKSFELKT HERRASFILG ELKPSKSLK RIIKNADIQA SSLQRVDLEK SIHKKELKST FDRIHNTEKY EYIGQPIIRH PVTGEIKEYG TILANLPQTQ R EVYDVDDE ...String:
MGSSHHHHHH SSGLVPRGSH MGQYFRVSGK YRGRENLPRE VNKMADNEFW LNKIRAFFHD PPDKSFELKT HERRASFILG ELKPSKSLK RIIKNADIQA SSLQRVDLEK SIHKKELKST FDRIHNTEKY EYIGQPIIRH PVTGEIKEYG TILANLPQTQ R EVYDVDDE GKEDYEEQFQ EILSRILKIE KKVFDDFKNR YSDPKDLYIS LWAFYAEKLK EALEEEFSAS FAEEFVNLPA YT LSPDHTL FDHADATSAI FGAEIDGKKP VLVLFKISPV QKFIADARKE KDLWAASHML STLTFKAISF IADKFGPDVV IYP HLRGNP FFHAWLHSKK IWEFSDSHSL KIASVPNKFL ALVGVSDEKE LNNLREGIRN EIESFLADLF DKLWNEVIVG ALEH SDALK HLGDKKEIHK EILLKRFTLT LSSLKIHDVD VSGSKEEAYE KVKDFVRSLG LPNAIESKYL QWLDMLGSVE ASNNR PTKY DLYSLYYEIL TVLNAIESTH FDKPAEPAGY KCTLCGEHLA IGGESREMME NVWGKIHKRW PSHLRSNERL CAVCAV KRF YPKFIETLDI FEGVGKVVPD IESVSEVAMC RRTKHGITWK EVYDYLRGLK NVDDEKLLGK LENLKHSVQT LINNVKS EL KSRKVYPEEF LEGLNRNFSN EILYSERLRD FNTLLDTLGF DAAKLGLDDV KNYETMISEL RERLSEVYKM LGEPPKYY A ILMMDGDEMG KLLSGEKLKT AEHYLHSAIL ERVSDALRVK AKTVRRLITP AAHSSISRAL KNFSVNHVPD VVRKGNGTL IYSGGDDVLV LLPVDTAFDV ATELAMTFST SWNGWEMLPG NKLSAGLLIV HYKHPLYDAL EKTRELLQKA KKLGRNAIAV GLLKRSGSY YESVVNFETL EDAKAVANLL VKEQVSPRII YELLNFADVI SKEFLHQLVK YEAVRHSIDK NLAEEFQSVF A RGHQGVRV ELEGNDEEIN KYISDGANLE TFLDKYEKAV DVIRKQVRGF LNLVKILYES IRGGAK

UniProtKB: GGDEF domain-containing protein

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Macromolecule #3: Type III-B CRISPR module-associated protein Cmr3

MacromoleculeName: Type III-B CRISPR module-associated protein Cmr3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Archaeoglobus fulgidus DSM 4304 (archaea)
Molecular weightTheoretical: 39.043328 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MILVMKPYDS LLFREPRPFD VNNHVARTIL PLPQTLAGAV RSAIYVKYGS KVKDLIGFGK EEPEFEILGH FFYRYDGKFE LLVESPHDV TQNLGLVKPH RIDKLGITIL MDSEGIKFRP FNGFLKFSGL IDYLQGRIAE DSVVERQKIF KKERRVGIAL T KAKVTKEE ...String:
MILVMKPYDS LLFREPRPFD VNNHVARTIL PLPQTLAGAV RSAIYVKYGS KVKDLIGFGK EEPEFEILGH FFYRYDGKFE LLVESPHDV TQNLGLVKPH RIDKLGITIL MDSEGIKFRP FNGFLKFSGL IDYLQGRIAE DSVVERQKIF KKERRVGIAL T KAKVTKEE HFYQVEMLRF SDDCGIAVWV EDGVDFDDEG ILGVGGERRF VKFEKREEPE CITNLRSKWK KIRDKINETG RL KIYLATP AILGAKGYSS KLDYDLLGDI GIERVRSVNF IGGKPVIFSG WDFVTRKPKP TRYAVPAGSV YFVEFEGEVK LDM PYLKLG KLTKLGYGLC FMGVW

UniProtKB: Type III-B CRISPR module-associated protein Cmr3

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Macromolecule #4: CRISPR type III-associated protein domain-containing protein

MacromoleculeName: CRISPR type III-associated protein domain-containing protein
type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Archaeoglobus fulgidus DSM 4304 (archaea)
Molecular weightTheoretical: 39.677582 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MFDMFEKAVV FGLYSITPVH AGSGAELSVI DLPIQRERHT GFPVIWGQSL KGVLRSRFRQ LELDEKIEVE SQKWKWKEKT KEVLKEKAD EFIKKVEERK RDPLLTEIVF GPATDGASEH AGAVSVGDAK ILLFPVRSAK GVFAFVTSPI VIQRLKEDFE L VSEIENDI ...String:
MFDMFEKAVV FGLYSITPVH AGSGAELSVI DLPIQRERHT GFPVIWGQSL KGVLRSRFRQ LELDEKIEVE SQKWKWKEKT KEVLKEKAD EFIKKVEERK RDPLLTEIVF GPATDGASEH AGAVSVGDAK ILLFPVRSAK GVFAFVTSPI VIQRLKEDFE L VSEIENDI ELKQILSRFK VELSNNETIA GNALILNGEN KVILEDIVLK VKSDSNVIEN LVEVLKTLFG DNFFGKPIES IK ERIAIVS DDVFKSFTRF STEIVARVRI DAEKGTVARG GLWYEEFLPS DTLMYSLIAV GSPKKENLPK EVDNTQKIVN VLK VTFNNA FLQIGGDETV GKGFVKVRAG VLTGGHKGN

UniProtKB: CRISPR type III-associated protein domain-containing protein

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Macromolecule #5: CRISPR system Cmr subunit Cmr5

MacromoleculeName: CRISPR system Cmr subunit Cmr5 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Archaeoglobus fulgidus DSM 4304 (archaea)
Molecular weightTheoretical: 17.407707 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MDIREIEQER ASFAFKVVSD IKDKYSQNKK VQGKYSSYAE KAPTIILNNG LGATLAFFLS KLEKPIDDVD YKSINPESFG NAENIAYAF LYKHLSTWLA EGNGKDSAFS GLTNGEDPLK YIMEKTAIDV AISTEEALSI LNWIKKFAKA MLEEEL

UniProtKB: CRISPR system Cmr subunit Cmr5

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Macromolecule #6: CRISPR type III-associated protein domain-containing protein

MacromoleculeName: CRISPR type III-associated protein domain-containing protein
type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Archaeoglobus fulgidus DSM 4304 (archaea)
Molecular weightTheoretical: 40.298773 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKSNVVVKYP VNSELANYAE KFWKKELAIY NLSLILNKMT PFVKRRSESY KSSLSAVKEI FKNVDDFQNF LNSVLRRSLD EYRVFFENY ERLFNSFSSK IFSMRTKSRL VVGLGDESVY ETSIRLHRNY GVPYIPGSAL KGVAKHYAFS ILARENGDEI L RIYESVKE ...String:
MKSNVVVKYP VNSELANYAE KFWKKELAIY NLSLILNKMT PFVKRRSESY KSSLSAVKEI FKNVDDFQNF LNSVLRRSLD EYRVFFENY ERLFNSFSSK IFSMRTKSRL VVGLGDESVY ETSIRLHRNY GVPYIPGSAL KGVAKHYAFS ILARENGDEI L RIYESVKE DLKARIAKRD KIKKNDVPED YYLTAAVIQE LFEKKFDELG AIRNTRVEIG DTVISVGDIV KIFGTQKEEG SV IFFDAFP TPEQLKDKPN LELDIMNPHY QPYYQHGEPP GDWHSPNPIF FLTVPAGVEF TFAVASRDLD DLAEKAEKLL KEA LKKFGV GAKTSLGYGR FDARDDRHHH HHH

UniProtKB: CRISPR type III-associated protein domain-containing protein

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Macromolecule #7: Target analog DNA

MacromoleculeName: Target analog DNA / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.451103 KDa
SequenceString:
(DT)(DG)(DC)(DT)(DC)(DT)(DC)(DA)(DG)(DC) (DC)(DG)(DC)(DA)(DA)(DG)(DG)(DA)(DC)(DC) (DG)(DC)(DA)(DT)(DA)(DC)(DT)(DA)(DC) (DA)(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

Concentration0.77 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMTris-HCl
100.0 mMNaCl
5.0 mMMgCl2
1.0 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Number grids imaged: 1 / Number real images: 2559 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 120000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.7.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.0) / Number images used: 81835
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.0)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 4.7.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-9x25:
Target analog-bound type III-B Cmr complex of Archaeoglobus fulgidus

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