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- EMDB-51149: CryoEM structure of Enterococcus italicus Csm-crRNA complex bound... -

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Basic information

Entry
Database: EMDB / ID: EMD-51149
TitleCryoEM structure of Enterococcus italicus Csm-crRNA complex bound to AMPNPP
Map dataP37J209 NUrefine map sharp
Sample
  • Complex: Csm-crRNA complex bound to AMPNPP with (Csm1)1(Csm2)2(Csm3)3(Csm4)1(Csm5)1 stoichiometry
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • Protein or peptide: CRISPR system Cms protein Csm2
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • Protein or peptide: CRISPR system Cms protein Csm4
    • RNA: RNA (28-MER)
    • Protein or peptide: CRISPR system Cms protein Csm5
  • Ligand: 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]adenosine
  • Ligand: MAGNESIUM ION
KeywordsType III-A CRISPR-Cas RNA-guided nuclease Endoribonuclease cyclic oligoadenylate synthetase HD nuclease / RNA BINDING PROTEIN
Function / homology
Function and homology information


exonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding / ATP binding
Similarity search - Function
: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / : / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 ...: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / : / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / : / 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
CRISPR system Cms protein Csm5 / CRISPR system Cms protein Csm4 / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm2 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
Similarity search - Component
Biological speciesEnterococcus italicus DSM 15952 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsJungfer K / Jinek M
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)820152European Union
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes.
Authors: Kenny Jungfer / Štefan Moravčík / Carmela Garcia-Doval / Anna Knörlein / Jonathan Hall / Martin Jinek /
Abstract: Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign ...Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign genetic elements, which triggers the generation of cyclic oligoadenylate (cOA) second messengers by the Cas10 subunit of the type III effector complex. In turn, cOAs bind and activate ancillary effector proteins to reinforce the host immune response. Type III systems utilize distinct cOAs, including cyclic tri- (cA3), tetra- (cA4) and hexa-adenylates (cA6). However, the molecular mechanisms dictating cOA product identity are poorly understood. Here we used cryoelectron microscopy to visualize the mechanism of cA6 biosynthesis by the Csm effector complex from Enterococcus italicus (EiCsm). We show that EiCsm synthesizes oligoadenylate nucleotides in 3'-5' direction using a set of conserved binding sites in the Cas10 Palm domains to determine the size of the nascent oligoadenylate chain. Our data also reveal that conformational dynamics induced by target RNA binding results in allosteric activation of Cas10 to trigger oligoadenylate synthesis. Mutations of a key structural element in Cas10 perturb cOA synthesis to favor cA3 and cA4 formation. Together, these results provide comprehensive insights into the dynamics of cOA synthesis in type III CRISPR-Cas systems and reveal key determinants of second messenger product selectivity, thereby illuminating potential avenues for their engineering.
History
DepositionJul 25, 2024-
Header (metadata) releaseJan 29, 2025-
Map releaseJan 29, 2025-
UpdateJan 29, 2025-
Current statusJan 29, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51149.png

Downloads & links

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Map

FileDownload / File: emd_51149.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationP37J209 NUrefine map sharp
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.064
Minimum - Maximum-0.49259397 - 0.7179152
Average (Standard dev.)-0.00004152167 (±0.012743237)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 312.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: P37J209 NUrefine map halfA

Fileemd_51149_half_map_1.map
AnnotationP37J209 NUrefine map halfA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: P37J209 NUrefine map halfB

Fileemd_51149_half_map_2.map
AnnotationP37J209 NUrefine map halfB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Csm-crRNA complex bound to AMPNPP with (Csm1)1(Csm2)2(Csm3)3(Csm4...

EntireName: Csm-crRNA complex bound to AMPNPP with (Csm1)1(Csm2)2(Csm3)3(Csm4)1(Csm5)1 stoichiometry
Components
  • Complex: Csm-crRNA complex bound to AMPNPP with (Csm1)1(Csm2)2(Csm3)3(Csm4)1(Csm5)1 stoichiometry
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • Protein or peptide: CRISPR system Cms protein Csm2
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • Protein or peptide: CRISPR system Cms protein Csm4
    • RNA: RNA (28-MER)
    • Protein or peptide: CRISPR system Cms protein Csm5
  • Ligand: 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]adenosine
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Csm-crRNA complex bound to AMPNPP with (Csm1)1(Csm2)2(Csm3)3(Csm4...

SupramoleculeName: Csm-crRNA complex bound to AMPNPP with (Csm1)1(Csm2)2(Csm3)3(Csm4)1(Csm5)1 stoichiometry
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4, #6, #5
Source (natural)Organism: Enterococcus italicus DSM 15952 (bacteria)
Molecular weightTheoretical: 260 KDa

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Macromolecule #1: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1...

MacromoleculeName: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
type: protein_or_peptide / ID: 1 / Details: N-term. 3C site & 10xHis tag / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Enterococcus italicus DSM 15952 (bacteria)
Molecular weightTheoretical: 88.294 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHHHH HLEVLFQGPS NKKLELMYGS LLHDIGKIVY RSNSVDFAKG THSKIGSQFL NKFKPFQLSG IVDSVSYHHY KELASSSLL DDSVAYITYI ADNIASGTDR RASEGDYEGE GNRQRFDKRA PLASIFNVVN SETKGLANYT YSFEKEQVYR Y PTDAKKEY ...String:
MHHHHHHHHH HLEVLFQGPS NKKLELMYGS LLHDIGKIVY RSNSVDFAKG THSKIGSQFL NKFKPFQLSG IVDSVSYHHY KELASSSLL DDSVAYITYI ADNIASGTDR RASEGDYEGE GNRQRFDKRA PLASIFNVVN SETKGLANYT YSFEKEQVYR Y PTDAKKEY TSSQYAALVN KMTDDLSNKL KVGPDSFSSL LQWTESLWSY IPSSTDTNQV MDVSLYDHSK ITCAIASCIY DY LTEMNCV NYRKELFSPY EKTKQFYQED VFLLVSLDMS GIQDFIYNIS GSKALKSLRS RSFYLETMLE SLVDDLLSDL ELS RANLLY TGGGHAYLLL PNTERARDVL ASFEGEMKEW FIKIFKTDLS VAIAYKACTG EDLMNSNGTY SDLWQTVSRK LSDK KAHKY SLNEIKLFNS TIHAGTQECK ECLRSDIDIS EDSLCKICEG IIAISNDLRD YSFFVVSPEG KVPLPRNRYL SVENQ DGAE RKIKMNKETR IYSKNQPFVG KQLVTNLWMC DYDFSTLNPE TKKQGIASYV NREVGIPRLG VLRADIDNLG TTFIKG IPE QYRSISRTAT LSRQLSMFFK FELSNILKGA RISVIYSGGD DLFLIGAWDD VISKALVLRK AFTRFSAGKL TFSAGIG MY PVKYPISKMA SETGVLEDLA KRGEKNQVAL WNDSKVFGWS QLEEQILKEK MIPLQEALTN SQEHGKSFLY KMLELLRN E DQINIARLAY LLARSSLSEE LTQSIFAWSQ NKQQKVELIT AIEYLVYQIR EAD

UniProtKB: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)

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Macromolecule #2: CRISPR system Cms protein Csm2

MacromoleculeName: CRISPR system Cms protein Csm2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Enterococcus italicus DSM 15952 (bacteria)
Molecular weightTheoretical: 16.523047 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MELAKTKTGE MIDLNFARKV VEENKRVKDN RGRQEIVLFN GLTTSKLRNL LELINHVYTK VYNSDDTTLS EDVRDELEYL KVKFAYESG REPAVRTFIE KTYVDKLVDV VLKKNTKKIF LDYCKYFEAL VAYAKFYRMG D

UniProtKB: CRISPR system Cms protein Csm2

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Macromolecule #3: CRISPR system Cms endoribonuclease Csm3

MacromoleculeName: CRISPR system Cms endoribonuclease Csm3 / type: protein_or_peptide / ID: 3 / Details: E32A / Number of copies: 3 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Enterococcus italicus DSM 15952 (bacteria)
Molecular weightTheoretical: 23.500777 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MYSKIRIVGK IDVLTGLHIG GGGETSMIGA IASPVVRDPY SRLPIIPGSS IKGKMRSLLA KHIGLIPGQK MHNQDAPEIL RLFGSSQKG AIQSSRLQIS DAFFSKASQE EFDKKDLAYT ETKFENTISR LTAVANPRQI ERVTRGASFD FHIIYNVENI N EVMADFEN ...String:
MYSKIRIVGK IDVLTGLHIG GGGETSMIGA IASPVVRDPY SRLPIIPGSS IKGKMRSLLA KHIGLIPGQK MHNQDAPEIL RLFGSSQKG AIQSSRLQIS DAFFSKASQE EFDKKDLAYT ETKFENTISR LTAVANPRQI ERVTRGASFD FHIIYNVENI N EVMADFEN IKTAIHLLEN DYLGGGGTRG NGRIRFVIDS IDTVVGDFDS SNLSIK

UniProtKB: CRISPR system Cms endoribonuclease Csm3

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Macromolecule #4: CRISPR system Cms protein Csm4

MacromoleculeName: CRISPR system Cms protein Csm4 / type: protein_or_peptide / ID: 4 / Details: Enterococcus italicus DSM 15952 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Enterococcus italicus DSM 15952 (bacteria)
Molecular weightTheoretical: 34.358059 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNQLVVKLVK LTFKSPVHFG MKRLSDSNHT IAADTLFSAL IIEALQQQLE LSHLLNNLVI TDLFPYNKTS YFLPKPLIRI EGKKGDESG YKAFKKLTYI PVENYSEYLR GEIDSLEASK IAESLNLGKA SLSTKVSLQA VDHNGESEPY SVGNFTFYPE S GLYFLAKG ...String:
MNQLVVKLVK LTFKSPVHFG MKRLSDSNHT IAADTLFSAL IIEALQQQLE LSHLLNNLVI TDLFPYNKTS YFLPKPLIRI EGKKGDESG YKAFKKLTYI PVENYSEYLR GEIDSLEASK IAESLNLGKA SLSTKVSLQA VDHNGESEPY SVGNFTFYPE S GLYFLAKG NADTIGQLEI LMHALQYSGI GGKRSAGYGQ FRCTIEDSGK FDSLLSQTGN IAILLSSAMA SDEELVDCLE DA RYLLKKR TGFVQSKTYA DQLVKKKDFY AFSAGSTFYQ KFNGKIFDVS DNGRHSVYRY AKAFWLEGKI

UniProtKB: CRISPR system Cms protein Csm4

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Macromolecule #5: CRISPR system Cms protein Csm5

MacromoleculeName: CRISPR system Cms protein Csm5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Enterococcus italicus DSM 15952 (bacteria)
Molecular weightTheoretical: 43.328676 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MIEKVYQVKL KVYGPVHIGS GKIIRKQEYI YDRRKSLAHI VDGPNLVKFL NKKGKFTAYL QYLNTTKERA DLYTFLRQEQ IDTNDWKTF VLYTERVNQG KIDMKDHNPY SRTSTNRRQV DKGMNDLHLF VRDGRGDLYI PGSSLKGALR TVLEGANQSA E AFHSLSIS ...String:
MIEKVYQVKL KVYGPVHIGS GKIIRKQEYI YDRRKSLAHI VDGPNLVKFL NKKGKFTAYL QYLNTTKERA DLYTFLRQEQ IDTNDWKTF VLYTERVNQG KIDMKDHNPY SRTSTNRRQV DKGMNDLHLF VRDGRGDLYI PGSSLKGALR TVLEGANQSA E AFHSLSIS DSLPIDPKNL AIYQKIDINK ELKPMPLYRE CVNVGTTVEF TMKINSDDWT IEKIEKQIQQ AYLQYWNKWF VG MVTTPGG KAFIKGGGLP SVLHAKHRPT VLFLGGGTGF PSKTTHYLQK PKEQAQKDIF AILQRRFRNV YGKMATVPKN VPM VLKGTV NDSTNKWYQQ GVCLLEFQPI GEALEVLFQG PGGGWSHPQF EKGGGWSHPQ FEK

UniProtKB: CRISPR system Cms protein Csm5

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Macromolecule #6: RNA (28-MER)

MacromoleculeName: RNA (28-MER) / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Enterococcus italicus DSM 15952 (bacteria)
Molecular weightTheoretical: 14.645835 KDa
SequenceString:
ACGAGAACAU GCGCGACAUU CCGAAGAACG CUGAAGCGCU GGGGG

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Macromolecule #7: 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phos...

MacromoleculeName: 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]adenosine
type: ligand / ID: 7 / Number of copies: 2 / Formula: ZAN
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ZAN:
5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]adenosine

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
350.0 mMKClpotassium chloride
10.0 mMMgClmagnesium chloride
1.0 mMDTTdithiothreitol
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 66.51 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 121528
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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