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Yorodumi- EMDB-9254: Cryo-EM structure of larger Csm-crRNA-target RNA ternary complex ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-9254 | |||||||||
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Title | Cryo-EM structure of larger Csm-crRNA-target RNA ternary complex in type III-A CRISPR-Cas system | |||||||||
Map data | Larger Csm-crRNA-target RNA ternary complex in type III-A CRISPR-Cas system | |||||||||
Sample |
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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 | |||||||||
Biological species | Thermococcus onnurineus (archaea) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||
Authors | Jia N / Wang C / Eng ET | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_9254.map.gz | 4.7 MB | EMDB map data format | |
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Header (meta data) | emd-9254-v30.xml emd-9254.xml | 17.4 KB 17.4 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_9254_fsc.xml | 9.1 KB | Display | FSC data file |
Images | emd_9254.png | 2.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-9254 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-9254 | HTTPS FTP |
-Validation report
Summary document | emd_9254_validation.pdf.gz | 353.4 KB | Display | EMDB validaton report |
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Full document | emd_9254_full_validation.pdf.gz | 353 KB | Display | |
Data in XML | emd_9254_validation.xml.gz | 10.9 KB | Display | |
Data in CIF | emd_9254_validation.cif.gz | 14.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-9254 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-9254 | HTTPS FTP |
-Related structure data
Related structure data | 6musMC 9253C 9255C 9256C 6muaC 6murC 6mutC 6muuC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_9254.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Larger Csm-crRNA-target RNA ternary complex in type III-A CRISPR-Cas system | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.089 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Csm-crRNA-target RNA larger complex
Entire | Name: Csm-crRNA-target RNA larger complex |
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Components |
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-Supramolecule #1: Csm-crRNA-target RNA larger complex
Supramolecule | Name: Csm-crRNA-target RNA larger complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7 |
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Source (natural) | Organism: Thermococcus onnurineus (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Molecular weight | Experimental: 300 KDa |
-Macromolecule #1: Uncharacterized protein Csm1
Macromolecule | Name: Uncharacterized protein Csm1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Thermococcus onnurineus (archaea) |
Molecular weight | Theoretical: 89.682547 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #2: Uncharacterized protein Csm2
Macromolecule | Name: Uncharacterized protein Csm2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Thermococcus onnurineus (archaea) |
Molecular weight | Theoretical: 21.210293 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: SMAYHQKHGG YGRGGYGRQD RPQVDASRLF GESPDVVGIK KMLEGKGKQW EAIQPYFDNV VREAKNFLEW SPNKRLANAV TVAAYLTSQ GLKTNQVRKI LDMARTTELK VKRGEGDIKD DLVKMRYLLA YTVGKATGQS KYSLDAFHRI LDPMLEVLMG S PKKENFEK FYDFLQAVVA YHKFFGGGD |
-Macromolecule #3: Uncharacterized protein Csm3
Macromolecule | Name: Uncharacterized protein Csm3 / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Thermococcus onnurineus (archaea) |
Molecular weight | Theoretical: 32.765002 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #4: Uncharacterized protein Csm4
Macromolecule | Name: Uncharacterized protein Csm4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Thermococcus onnurineus (archaea) |
Molecular weight | Theoretical: 32.345061 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #7: Uncharacterized protein Csm5
Macromolecule | Name: Uncharacterized protein Csm5 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Thermococcus onnurineus (archaea) |
Molecular weight | Theoretical: 46.091016 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #5: RNA (33-MER)
Macromolecule | Name: RNA (33-MER) / type: rna / ID: 5 / Number of copies: 1 |
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Source (natural) | Organism: Thermococcus onnurineus (archaea) |
Molecular weight | Theoretical: 12.463438 KDa |
Sequence | String: GUGGAAAGGC GGGCAGAGGC GGUUUGCGUA UUGGGCGC |
-Macromolecule #6: RNA (25-MER)
Macromolecule | Name: RNA (25-MER) / type: rna / ID: 6 / Number of copies: 1 |
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Source (natural) | Organism: Thermococcus onnurineus (archaea) |
Molecular weight | Theoretical: 12.750658 KDa |
Sequence | String: CCCUGGCGCC CAAUACGCAA ACCGCCUCUG CCCGCGGGCG |
-Macromolecule #8: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 8 / Number of copies: 4 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 8.8 / Component - Formula: Tris / Details: 20 mM Tris-HCl, pH 8.8, 250 mM NaCl, 2 mM DTT |
Grid | Details: unspecified |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.35 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |