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- EMDB-9255: Cryo-EM structure of ternary Csm-crRNA-target RNA with anti-tag s... -

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Basic information

Entry
Database: EMDB / ID: EMD-9255
TitleCryo-EM structure of ternary Csm-crRNA-target RNA with anti-tag sequence complex in type III-A CRISPR-Cas system
Map dataTernary Csm-crRNA-target RNA with anti-tag sequence complex in type III-A CRISPR-Cas system
Sample
  • Complex: ternary Csm-crRNA-target RNA with anti-tag sequence complex
    • Protein or peptide: Uncharacterized protein Csm1
    • Protein or peptide: Uncharacterized protein Csm2
    • Protein or peptide: Uncharacterized protein Csm3
    • Protein or peptide: Uncharacterized protein Csm4
    • RNA: RNA (5'-R(*GP*UP*GP*GP*AP*AP*AP*GP*GP*CP*GP*GP*GP*CP*AP*GP*AP*GP*GP*C)-3')
    • RNA: RNA (5'-R(P*CP*CP*UP*CP*UP*GP*CP*CP*CP*GP*CP*CP*UP*UP*UP*CP*CP*AP*C)-3')
    • Protein or peptide: Uncharacterized protein Csm5
  • Ligand: ZINC ION
Keywordscryo-EM structure / ternary Csm-crRNA-target RNA with anti-tag sequence complex / Type III CRISPR-Cas system / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


exonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / defense response to virus / endonuclease activity / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsJia N / Wang C
CitationJournal: Mol Cell / Year: 2019
Title: Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity.
Authors: Ning Jia / Charlie Y Mo / Chongyuan Wang / Edward T Eng / Luciano A Marraffini / Dinshaw J Patel /
Abstract: Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on ...Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on cryo-EM structures of Thermococcus onnurineus Csm binary, Csm-target RNA and Csm-target RNA ternary complexes in the 3.1 Å range. The topological features of the crRNA 5'-repeat tag explains the 5'-ruler mechanism for defining target cleavage sites, with accessibility of positions -2 to -5 within the 5'-repeat serving as sensors for avoidance of autoimmunity. The Csm3 thumb elements introduce periodic kinks in the crRNA-target RNA duplex, facilitating cleavage of the target RNA with 6-nt periodicity. Key Glu residues within a Csm1 loop segment of Csm adopt a proposed autoinhibitory conformation suggestive of DNase activity regulation. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into Csm complex assembly, mechanisms underlying RNA targeting and site-specific periodic cleavage, regulation of DNase cleavage activity, and autoimmunity suppression.
History
DepositionOct 23, 2018-
Header (metadata) releaseNov 14, 2018-
Map releaseDec 12, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6mut
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9255.png

Downloads & links

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Map

FileDownload / File: emd_9255.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTernary Csm-crRNA-target RNA with anti-tag sequence complex in type III-A CRISPR-Cas system
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: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.14048749 - 0.26415032
Average (Standard dev.)0.00024085725 (±0.0050697285)
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
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1400.2640.000

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Supplemental data

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Sample components

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Entire : ternary Csm-crRNA-target RNA with anti-tag sequence complex

EntireName: ternary Csm-crRNA-target RNA with anti-tag sequence complex
Components
  • Complex: ternary Csm-crRNA-target RNA with anti-tag sequence complex
    • Protein or peptide: Uncharacterized protein Csm1
    • Protein or peptide: Uncharacterized protein Csm2
    • Protein or peptide: Uncharacterized protein Csm3
    • Protein or peptide: Uncharacterized protein Csm4
    • RNA: RNA (5'-R(*GP*UP*GP*GP*AP*AP*AP*GP*GP*CP*GP*GP*GP*CP*AP*GP*AP*GP*GP*C)-3')
    • RNA: RNA (5'-R(P*CP*CP*UP*CP*UP*GP*CP*CP*CP*GP*CP*CP*UP*UP*UP*CP*CP*AP*C)-3')
    • Protein or peptide: Uncharacterized protein Csm5
  • Ligand: ZINC ION

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Supramolecule #1: ternary Csm-crRNA-target RNA with anti-tag sequence complex

SupramoleculeName: ternary Csm-crRNA-target RNA with anti-tag sequence complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Thermococcus onnurineus (archaea)
Molecular weightTheoretical: 250 KDa

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Macromolecule #1: Uncharacterized protein Csm1

MacromoleculeName: Uncharacterized protein Csm1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus onnurineus (archaea)
Molecular weightTheoretical: 89.750602 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SQDPMEIDEL TALGGLLHDI GKPVQRAGLY SGDHSTQGAR FLRDLAENTG RAEYELLSLF SEFHHKGHMK NDELMIRRI KELSPERFGL TMEDVLNALW IVYEADNLAS GEREEGQPQA SRPLYSVFNP GKAYPWAELD FEKELPVPGD V FSIRSQDY ...String:
MGSSHHHHHH SQDPMEIDEL TALGGLLHDI 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: Uncharacterized protein Csm2

MacromoleculeName: Uncharacterized protein Csm2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus onnurineus (archaea)
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: Uncharacterized protein Csm3

MacromoleculeName: Uncharacterized protein Csm3 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus onnurineus (archaea)
Molecular weightTheoretical: 32.765002 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SMDRRFYGKI VIKGKIKAVT GLHIGSQRDI SEIGGIANPV IKDPHTGLPY IPGSSLKGRL RSLFEILVNS RLGEWREKYP SLANYSPGS CRPDNQENCG KFFNRKINRG WIHVCPDYET ALACPVCRLF GASGKESNFP SRIIVRDAFL TKEWEEKWRA G EAITEAKI ...String:
SMDRRFYGKI VIKGKIKAVT GLHIGSQRDI SEIGGIANPV 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: Uncharacterized protein Csm4

MacromoleculeName: Uncharacterized protein Csm4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus onnurineus (archaea)
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: Uncharacterized protein Csm5

MacromoleculeName: Uncharacterized protein Csm5 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus onnurineus (archaea)
Molecular weightTheoretical: 46.091016 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTERTLKVLS PLHIGTGNEL TPVDIYPREN IIHVLDTERL VNDLMNLGVE LNEILALLKN PPGDAYIWKG YIEEFHLDPS DYSIYTLKI HGKIGRKSMQ IKEFIKLNGR PYIPGSSLKG AIRTAVLYKA LKECGDARAV MRVVSKVNGD VARDIGRSED V LDYYMSFL ...String:
MTERTLKVLS PLHIGTGNEL TPVDIYPREN IIHVLDTERL VNDLMNLGVE LNEILALLKN PPGDAYIWKG YIEEFHLDPS DYSIYTLKI HGKIGRKSMQ IKEFIKLNGR PYIPGSSLKG AIRTAVLYKA LKECGDARAV MRVVSKVNGD VARDIGRSED V LDYYMSFL SRARIDRKRA DDLLEAIVFG MEPDRRSKIR YEPKRDPMKA LIVRDSKPVG RKHLAVYHVE VIGNPQPIPI WV EAIEPGA ATDVEIHVDT EALRLNADYF NGLLWECLKE RGEPGEVFED FLWEAVDEFY TAVMKYETIE VQKFGRYTSQ VRS FYASLE DHSGHVLRLG WGSGWLAMTI GLLLVEKGYK WENVRKKLGL GKKPGGSGFS REFPKTRRLA DGMPMGWVVL EHHH HHH

UniProtKB: CRISPR system Cms protein Csm5

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Macromolecule #5: RNA (5'-R(*GP*UP*GP*GP*AP*AP*AP*GP*GP*CP*GP*GP*GP*CP*AP*GP*AP*GP*...

MacromoleculeName: RNA (5'-R(*GP*UP*GP*GP*AP*AP*AP*GP*GP*CP*GP*GP*GP*CP*AP*GP*AP*GP*GP*C)-3')
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 (5'-R(P*CP*CP*UP*CP*UP*GP*CP*CP*CP*GP*CP*CP*UP*UP*UP*CP*CP*AP...

MacromoleculeName: RNA (5'-R(P*CP*CP*UP*CP*UP*GP*CP*CP*CP*GP*CP*CP*UP*UP*UP*CP*CP*AP*C)-3')
type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Thermococcus onnurineus (archaea)
Molecular weightTheoretical: 14.223495 KDa
SequenceString:
CCCUGGCGCC CAAUACGCAA ACCGCCUCUG CCCGCCUUUC CACGG

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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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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
GridDetails: unspecified
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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 57170
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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