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- EMDB-0640: Cryo-EM structure of Csm-crRNA-target RNA ternary complex in comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-0640
TitleCryo-EM structure of Csm-crRNA-target RNA ternary complex in complex with AMPPNP in type III-A CRISPR-Cas system
Map data
Sample
  • Complex: Csm-crRNA-target RNA complex
    • Protein or peptide: Csm1
    • Protein or peptide: Csm2
    • Protein or peptide: Csm3
    • Protein or peptide: Csm4
    • RNA: RNA (38-MER)
    • RNA: RNA (40-MER)
    • Protein or peptide: Csm5
  • Ligand: ZINC ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MANGANESE (II) ION
Keywordscryo-EM structure / Csm-crRNA-target RNA ternary complex in complex with AMPPNP / Type III CRISPR-Cas systerm / IMMUNE SYSTEM / immune system-rna complex
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 / identical protein binding
Similarity search - Function
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-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 / HD domain profile. / GGDEF domain profile. / GGDEF domain / HD domain / HD domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm2 / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm5
Similarity search - Component
Biological speciesThermococcus onnurineus (archaea) / Thermococcus onnurineus (strain NA1) (archaea) / Thermococcus onnurineus NA1 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsJia N / Patel DJ
CitationJournal: Mol Cell / Year: 2019
Title: Second Messenger cA Formation within the Composite Csm1 Palm Pocket of Type III-A CRISPR-Cas Csm Complex and Its Release Path.
Authors: Ning Jia / Roger Jones / George Sukenick / Dinshaw J Patel /
Abstract: Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA) formation from ATP subunits positioned within the composite pair ...Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cA second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppA), and products (cA), to decipher mechanistic aspects of cA formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppA intermediates and subsequent cA formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cA signaling pathway.
History
DepositionMar 7, 2019-
Header (metadata) releaseMay 1, 2019-
Map releaseJul 31, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6o7e
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0640.png

Downloads & links

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Map

FileDownload / File: emd_0640.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 256 pix.
= 278.784 Å		1.09 Å/pix.
x 256 pix.
= 278.784 Å		1.09 Å/pix.
x 256 pix.
= 278.784 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.089 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.16742365 - 0.3156488
Average (Standard dev.)0.0002805735 (±0.005765763)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 278.784 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0891.0891.089
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z278.784278.784278.784
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1670.3160.000

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Supplemental data

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Sample components

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Entire : Csm-crRNA-target RNA complex

EntireName: Csm-crRNA-target RNA complex
Components
  • Complex: Csm-crRNA-target RNA complex
    • Protein or peptide: Csm1
    • Protein or peptide: Csm2
    • Protein or peptide: Csm3
    • Protein or peptide: Csm4
    • RNA: RNA (38-MER)
    • RNA: RNA (40-MER)
    • Protein or peptide: Csm5
  • Ligand: ZINC ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Csm-crRNA-target RNA complex

SupramoleculeName: Csm-crRNA-target RNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Thermococcus onnurineus (archaea)
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: Csm1

MacromoleculeName: Csm1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Thermococcus onnurineus (strain NA1) (archaea) / Strain: NA1
Molecular weightTheoretical: 89.682547 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SQDPMEIDEL TALGGLLANI GKPVQRAGLY SGDHSTQGAR FLRDLAENTG RAEYELLSLF SEFHHKGHMK NDELMIRRI KELSPERFGL TMEDVLNALW IVYEADNLAS GEREEGQPQA SRPLYSVFNP GKAYPWAELD FEKELPVPGD V FSIRSQDY ...String:
MGSSHHHHHH SQDPMEIDEL TALGGLLANI GKPVQRAGLY SGDHSTQGAR FLRDLAENTG RAEYELLSLF SEFHHKGHMK NDELMIRRI KELSPERFGL TMEDVLNALW IVYEADNLAS GEREEGQPQA SRPLYSVFNP GKAYPWAELD FEKELPVPGD V FSIRSQDY RELVKRLWEE LSKAKLRSDR LLPVLEKYLT FVSSVTSEGN IISLYDHMRM TSAIALAMLR AGCTAEDVRS GR CRKEKRF LLIEGDFSGI QDFIYRVSGK GTLKYLRARS AYLELIGWDV VLEILSRLGL TRANVVFNAG GHFMIIAQNT PDA VKELEE IRAKAVEWLY REFESDLYLA IEWEPVSGRE FGREGGKNLF AEARKRLKHK LTVRKLKRFG EIKGLFEHGH TERL AECPV CGRELPEGKL EPSASDPETK VCPTCNRLVS LGGNLPKLLG FGRTAKNDAG VLVEGPFSGF VPYLQGGRPV GEQIL VKNT LNPGEIPESA QFVPYFVADY FKKDPKGGVA TFEELSMAST GTRRLGVMKG DVDRLGEFFS SMDSPSKLAT ASRFMD YFF KGYIGAIIEG KFGYIIGDVP SLRDWPEEPD IVVVYAGGDD FFIVGAWDQI FELAFRVRRA FNAYTGGKLT LSVGLGY FD ERTPIYRMAD VVSERLDTAK DEGRNRVFVV GRSRPLDGKH KLSYEWNHYE ELWRTYAPRI YAGNGRLKGK LESKKGLL W KLLEIRELYV RDPNDVRWAY LTAYLLGRHG LSDLFPELVG IDTKAVERKE PQPVYWVDGV LKIVLMAVRR

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

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Macromolecule #2: Csm2

MacromoleculeName: Csm2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus onnurineus (strain NA1) (archaea) / Strain: NA1
Molecular weightTheoretical: 21.210293 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SMAYHQKHGG YGRGGYGRQD RPQVDASRLF GESPDVVGIK KMLEGKGKQW EAIQPYFDNV VREAKNFLEW SPNKRLANAV TVAAYLTSQ GLKTNQVRKI LDMARTTELK VKRGEGDIKD DLVKMRYLLA YTVGKATGQS KYSLDAFHRI LDPMLEVLMG S PKKENFEK FYDFLQAVVA YHKFFGGGD

UniProtKB: CRISPR system Cms protein Csm2

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Macromolecule #3: Csm3

MacromoleculeName: Csm3 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus onnurineus (strain NA1) (archaea) / Strain: NA1
Molecular weightTheoretical: 32.809012 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SMDRRFYGKI VIKGKIKAVT GLHIGSQRDI SEIGGIDNPV IKDPHTGLPY IPGSSLKGRL RSLFEILVNS RLGEWREKYP SLANYSPGS CRPDNQENCG KFFNRKINRG WIHVCPDYET ALACPVCRLF GASGKESNFP SRIIVRDAFL TKEWEEKWRA G EAITEAKI ...String:
SMDRRFYGKI VIKGKIKAVT GLHIGSQRDI SEIGGIDNPV IKDPHTGLPY IPGSSLKGRL RSLFEILVNS RLGEWREKYP SLANYSPGS CRPDNQENCG KFFNRKINRG WIHVCPDYET ALACPVCRLF GASGKESNFP SRIIVRDAFL TKEWEEKWRA G EAITEAKI EVGIDRVTSQ ANPRTNERVV AGAEFEFEII YNVENTTHWR DDIKNLLTAM ALLEDSYLGG SGSRGYGKVK FI FDSFEFR PLDYYRTGKD EDIVSIDARE KSVSDILSGF DSLFSEVEGK LEAG

UniProtKB: CRISPR system Cms endoribonuclease Csm3

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Macromolecule #4: Csm4

MacromoleculeName: Csm4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus onnurineus (strain NA1) (archaea) / Strain: NA1
Molecular weightTheoretical: 32.345061 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPKFIAVKLI PKGPFRDIPR ADTLFGAIGN AISAIHGQSA VEELVDAFVG GARISSAFPY SGDTYYLPKP LSVEPALEGI LTGLDEEER YTTAKRLRKA KYLDLKNFEL ALRLRPFTIP EEIPYARVDV PRVVLDRVTQ DSSIYFWEEI RFREKSGVYF L YSGPREVF ...String:
MPKFIAVKLI PKGPFRDIPR ADTLFGAIGN AISAIHGQSA VEELVDAFVG GARISSAFPY SGDTYYLPKP LSVEPALEGI LTGLDEEER YTTAKRLRKA KYLDLKNFEL ALRLRPFTIP EEIPYARVDV PRVVLDRVTQ DSSIYFWEEI RFREKSGVYF L YSGPREVF DGYIAPAMRF LGDTGIGGKS TWGAGLFEVE FHEMKIDAPG SEYSVTLSNA LPTKTPVLWR LLRKGGWSFG RR KPRMTFI AEGSIVKNDP GGMERLELGL SHEVYVYGLT FPLGVELPEG LE

UniProtKB: CRISPR system Cms protein Csm4

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Macromolecule #7: Csm5

MacromoleculeName: Csm5 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus onnurineus NA1 (archaea) / Strain: NA1
Molecular weightTheoretical: 40.58841 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTERTLKVLS PLHIGTGNEL TPVDIYPREN IIHVLDTER(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
MTERTLKVLS PLHIGTGNEL TPVDIYPREN IIHVLDTER(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)RKSMQIKEF IKLNGRPYIP GSSLKGAIRT AVLYKA LKE C(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)IRY EPKRDPMKAL IV RDSKPVG RKHLAVYHVE VIGNPQPIPI WVEAIEPGAA TDVEIHVDTE ALRLNADYFN GLLWECLKER GEPGEVFEDF LWE AVDEFY TAVMKYETIE VQKFGRYTSQ VRSFYASLED HSGHVLRLGW GSGWLAMTIG LLLVEKGYKW ENVRKKLGLG KKPG GSGFS REFPKTRRLA DGMPMGWVVL EHHHHHH

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Macromolecule #5: RNA (38-MER)

MacromoleculeName: RNA (38-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Thermococcus onnurineus (archaea)
Molecular weightTheoretical: 12.463438 KDa
SequenceString:
GUGGAAAGGC GGGCAGAGGC GGUUUGCGUA UUGGGCGC

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Macromolecule #6: RNA (40-MER)

MacromoleculeName: RNA (40-MER) / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Thermococcus onnurineus (archaea)
Molecular weightTheoretical: 12.750658 KDa
SequenceString:
CCCUGGCGCC CAAUACGCAA ACCGCCUCUG CCCGCGGGCG

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 3 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #9: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 9 / Number of copies: 2 / 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 #10: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 10 / Number of copies: 3 / Formula: MN
Molecular weightTheoretical: 54.938 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

Concentration0.5 mg/mL
BufferpH: 8.8 / Component - Formula: Tris / Details: 20 mM Tris-HCl, pH 8.8, 250 mM NaCl, 2 mM DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.35 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 110194
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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