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- EMDB-40251: CRISPR-Cas type III-D effector complex bound to self-target RNA i... -

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Basic information

Entry
Database: EMDB / ID: EMD-40251
TitleCRISPR-Cas type III-D effector complex bound to self-target RNA in a post-cleavage state
Map dataCRISPR-Cas type III-D complex in a post-RNA cleavage state
Sample
  • Complex: CRISPR-Cas type III-D effector complex
    • Protein or peptide: Cas7-Cas5-Cas11
    • Protein or peptide: TIGR03984 family CRISPR-associated protein
    • Protein or peptide: Cas10
    • Protein or peptide: Cas7-2x
    • Protein or peptide: TIGR03986 family CRISPR-associated RAMP protein
    • RNA: CRISPR RNA
  • RNA: Self-target RNA
KeywordsCRISPR / CRISPR-Cas / type III / complex / RNA / crRNA / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


defense response to virus / RNA binding
Similarity search - Function
CRISPR-associated protein A791736 / CRISPR-associated RAMP BGP1436 / Cas10/Cmr2, second palm domain / : / CRISPR type III-associated protein / RAMP superfamily / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
GGDEF domain-containing protein / DUF324 domain-containing protein / DUF324 domain-containing protein / TIGR03984 family CRISPR-associated protein / DUF324 domain-containing protein
Similarity search - Component
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsSchwartz EA / Taylor DW
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Nat Commun / Year: 2024
Title: RNA targeting and cleavage by the type III-Dv CRISPR effector complex.
Authors: Evan A Schwartz / Jack P K Bravo / Mohd Ahsan / Luis A Macias / Caitlyn L McCafferty / Tyler L Dangerfield / Jada N Walker / Jennifer S Brodbelt / Giulia Palermo / Peter C Fineran / Robert D ...Authors: Evan A Schwartz / Jack P K Bravo / Mohd Ahsan / Luis A Macias / Caitlyn L McCafferty / Tyler L Dangerfield / Jada N Walker / Jennifer S Brodbelt / Giulia Palermo / Peter C Fineran / Robert D Fagerlund / David W Taylor /
Abstract: CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular ...CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.
History
DepositionMar 30, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40251.png

Downloads & links

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Map

FileDownload / File: emd_40251.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCRISPR-Cas type III-D complex in a post-RNA cleavage state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 512 pix.
= 426.598 Å		0.83 Å/pix.
x 512 pix.
= 426.598 Å		0.83 Å/pix.
x 512 pix.
= 426.598 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.505
Minimum - Maximum-1.0313022 - 2.0480864
Average (Standard dev.)-0.0006020148 (±0.04775922)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 426.5984 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map B

Fileemd_40251_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_40251_half_map_2.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CRISPR-Cas type III-D effector complex

EntireName: CRISPR-Cas type III-D effector complex
Components
  • Complex: CRISPR-Cas type III-D effector complex
    • Protein or peptide: Cas7-Cas5-Cas11
    • Protein or peptide: TIGR03984 family CRISPR-associated protein
    • Protein or peptide: Cas10
    • Protein or peptide: Cas7-2x
    • Protein or peptide: TIGR03986 family CRISPR-associated RAMP protein
    • RNA: CRISPR RNA
  • RNA: Self-target RNA

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Supramolecule #1: CRISPR-Cas type III-D effector complex

SupramoleculeName: CRISPR-Cas type III-D effector complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 332 KDa

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Macromolecule #1: Cas7-Cas5-Cas11

MacromoleculeName: Cas7-Cas5-Cas11 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 87.558938 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRGIEITITM QSDWHVGTGM GRGELDSVVQ RDGDNLPYIP GKTLTGILRD SCEQVALGLD NGQTRGLWHG WINFIFGDQP ALAQGAIEP EPRPALIAIG SAHLDPKLKA AFQGKKQLQE AIAFMKPGVA IDAITGTAKK DFLRFEEVVR LGAKLTAEVE L NLPDNLSE ...String:
MRGIEITITM QSDWHVGTGM GRGELDSVVQ RDGDNLPYIP GKTLTGILRD SCEQVALGLD NGQTRGLWHG WINFIFGDQP ALAQGAIEP EPRPALIAIG SAHLDPKLKA AFQGKKQLQE AIAFMKPGVA IDAITGTAKK DFLRFEEVVR LGAKLTAEVE L NLPDNLSE TNKKVIAGIL ASGAKLTERL GGKRRRGNGR CELKFSGYSD QQIQWLKDNY QSVDQPPKYQ QNKLQSAGDN PE QQPPWHI IPLTIKTLSP VVLPARTVGN VVECLDYIPG RYLLGYIHKT LGEYFDVSQA IAAGDLIITN ATIKIDGKAG RAT PFCLFG EKLDGGLGKG KGVYNRFQES EPDGIQLKGE RGGYVGQFEQ EQRNLPNTGK INSELFTHNT IQDDVQRPTS DVGG VYSYE AIIAGQTFVA ELRLPDSLVK QITSKNKNWQ AQLKATIRIG QSKKDQYGKI EVTSGNSADL PKPTGNNKTL SIWFL SDIL LRGDRLNFNA TPDDLKKYLE NALDIKLKER SDNDLICIAL RSQRTESWQV RWGLPRPSLV GWQAGSCLIY DIESGT VNA EKLQELMITG IGDRCTEGYG QIGFNDPLLS ASLGKLTAKP KASNNQSQNS QSNPLPTNHP TQDYARLIEK AAWREAI QN KALALASSRA KREEILGIKI MGKDSQPTMT QLGGFRSVLK RLHSRNNRDI VTGYLTALEQ VSNRKEKWSN TSQGLTKI R NLVTQENLIW NHLDIDFSPL TITQNGVNQL KSELWAEAVR TLVDAIIRGH KRDLEKAQEN ESNQQSQGAA

UniProtKB: DUF324 domain-containing protein

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Macromolecule #2: TIGR03984 family CRISPR-associated protein

MacromoleculeName: TIGR03984 family CRISPR-associated protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 21.899844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPAGGRLMKN LYHYHQYEIT LESAVDSCKN HLQAAIGLLY SPQKCELVKL DNSGKLVDSY NRLKFNNLGV FEARFFNLNC ELRWVNESN GNGTAVLLSE SDITLTGFEK GLQEFITAID QQYLLWGEPA KHPPNADGWQ RLAEARIGKL DIPLDNPLKP K DRVFLTSE ...String:
MPAGGRLMKN LYHYHQYEIT LESAVDSCKN HLQAAIGLLY SPQKCELVKL DNSGKLVDSY NRLKFNNLGV FEARFFNLNC ELRWVNESN GNGTAVLLSE SDITLTGFEK GLQEFITAID QQYLLWGEPA KHPPNADGWQ RLAEARIGKL DIPLDNPLKP K DRVFLTSE EYIAEVDDFG NCAVIDERLI KLEVK

UniProtKB: TIGR03984 family CRISPR-associated protein

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Macromolecule #3: Cas10

MacromoleculeName: Cas10 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 64.335309 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAHHHHHHVG TENLYFQGFL VLIETSGNQH FIFSTNKLRE NIGASELTYL ATTEILFQGV DRVFQTNYYD QWSDTNSLNF LADSKLNPA IDDPKNNADI EILLATSGKA IALVKEEGKA KQLIKEVTKQ ALINAPGLEI GGIYVNCNWQ DKLGVAKAVK E AHKQFEVN ...String:
MAHHHHHHVG TENLYFQGFL VLIETSGNQH FIFSTNKLRE NIGASELTYL ATTEILFQGV DRVFQTNYYD QWSDTNSLNF LADSKLNPA IDDPKNNADI EILLATSGKA IALVKEEGKA KQLIKEVTKQ ALINAPGLEI GGIYVNCNWQ DKLGVAKAVK E AHKQFEVN RAKRAGANGR FLRLPIAAGC SVSELPASDF DYNADGDKIP VSTVSKVKRE TAKSAKKRLR SVDGRLVNDL AQ LEKSFDE LDWLAVVHAD GNGLGQILLS LEKYIGEQTN RNYIDKYRRL SLALDNCTIN AFKMAIAVFK EDSKKIDLPI VPL ILGGDD LTVICRGDYA LEFTREFLEA FEGQTETHDD IKVIAQKAFG VDRLSACAGI SIIKPHFPFS VAYTLAERLI KSAK EVKQK VTVTNSSPIT PFPCSAIDFH ILYDSSGIDF DRIREKLRPE DNTELYNRPY VVTAAENLSQ AQGYEWSQAH SLQTL ADRV SYLRSEDGEG KSALPSSQSH ALRTALYLEK NEADAQYSLI SQRYKILKNF AEDGENKSLF HLENGKYVTR FLDALD AKD FFANANHKNQ GE

UniProtKB: GGDEF domain-containing protein

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Macromolecule #4: Cas7-2x

MacromoleculeName: Cas7-2x / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 56.820832 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MARKVTTRWK ITGTLIAETP LHIGGVGGDA DTDLALAVNG AGEYYVPGTS LAGALRGWMT QLLNNDESQI KDLWGDHLDA KRGASFVIV DDAVIHIPNN ADVEIREGVG IDRHFGTAAN GFKYSRAVIP KGSKFKLPLT FDSQDDGLPN ALIQLLCALE A GDIRLGAA ...String:
MARKVTTRWK ITGTLIAETP LHIGGVGGDA DTDLALAVNG AGEYYVPGTS LAGALRGWMT QLLNNDESQI KDLWGDHLDA KRGASFVIV DDAVIHIPNN ADVEIREGVG IDRHFGTAAN GFKYSRAVIP KGSKFKLPLT FDSQDDGLPN ALIQLLCALE A GDIRLGAA KTRGLGRIKL DDLKLKSFAL DKPEGIFSAL LDQGKKLDWN QLKANVTYQS PPYLGISITW NPKDPVMVKA EG DGLAIDI LPLVSQVGSD VRFVIPGSSI KGILRTQAER IIRTICQSNG SEKNFLEQLR INLVNELFGS ASLSQKQNGK DID LGKIGA LAVNDCFSSL SMTPDQWKAV ENATEMTGNL QPALKQATGY PNNISQAYKV LQPAMHVAVD RWTGGAAEGM LYSV LEPIG VTWEPIQVHL DIARLKNYYH GKEEKLKPAI ALLLLVLRDL ANKKIPVGYG TNRGMGTITV SQITLNGKAL PTELE PLNK TMTCPNLTDL DEAFRQDLST AWKEWIADPI DLCQQEAA

UniProtKB: DUF324 domain-containing protein

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Macromolecule #5: TIGR03986 family CRISPR-associated RAMP protein

MacromoleculeName: TIGR03986 family CRISPR-associated RAMP protein / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 90.334984 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTVGTLGVVG SAKNLKLQLS FINTRQQYVQ ITLFERNSFK VAEEEFSTEL VEIIKTALPT LKNKKVEFEE DGDQIKQIRE KGQAWVGAA EQIAPYVLPS GNITETPRNV NASNFHNPYN FVPALPRDGI TGDLGDCAPA GHSYYHGDKY SGRIAVKLTT V TPLLIPDA ...String:
MTVGTLGVVG SAKNLKLQLS FINTRQQYVQ ITLFERNSFK VAEEEFSTEL VEIIKTALPT LKNKKVEFEE DGDQIKQIRE KGQAWVGAA EQIAPYVLPS GNITETPRNV NASNFHNPYN FVPALPRDGI TGDLGDCAPA GHSYYHGDKY SGRIAVKLTT V TPLLIPDA SKEEINNNHK TYPVRIGKDG KPYLPPTSIK GMLRSAYEAV TNSRLAVFED HDSRLAYRMP ATMGLQMVPA RI EGDNIVL YPGTSRIGNN GRPANNDPMY AAWLPYYQNR IAYDGSRDYQ MAEHGDHVRF WAERYTRGNF CYWRVRQIAR HNQ NLGNRP ERGRNYGQHH STGVIEQFEG FVYKTNKNIG NKHDERVFII DRESIEIPLS RDLRRKWREL ITSYQEIHKK EVDR GDTGP SAVNGAVWSR QIIADESERN LSDGTLCYAH VKKEDGQYKI LNLYPVMITR GLYEIAPVDL LDETLKPATD KKQLS PADR VFGWVNQRGN GCYKGQLRIH SVTCQHDDAI DDFGNQNFSV PLAILGQPKP EQARFYCADD RKGIPLEDGY DRDDGY SDS EQGLRGRKVY PHHKGLPNGY WSNPTEDRSQ QAIQGHYQEY RRPKKDGLEQ RDDQNRSVKG WVKPLTEFTF EIDVTNL SE VELGALLWLL TLPDLHFHRL GGGKPLGFGS VRLDIDPDKT DLRNGAGWRD YYGSLLETSQ PDFTTLISQW INAFQTAV K EEYGSSSFDQ VTFIKASGQS LQGFHDNASI HYPRSTPEPK PDGEAFKWFV ANEKGRRLAL PALEKSQSFP IKPS

UniProtKB: DUF324 domain-containing protein

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Macromolecule #6: CRISPR RNA

MacromoleculeName: CRISPR RNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 11.927167 KDa
SequenceString:
ACUGAAACUG UAGUAGAACC AAUCGGGGUC GUCAAUA

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Macromolecule #7: Self-target RNA

MacromoleculeName: Self-target RNA / type: rna / ID: 7 / Number of copies: 1
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 19.265455 KDa
SequenceString:
CAUGACGGAU CGCGGGAGUU AUUGACGACC CCGAUUGGUU CUACUACAAA CGUGAUACUA

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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

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMHEPES-NaOH
100.0 mMPotassium ChlorideKCl
5.0 %Glycerol
1.0 mMDTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsParticles were monodisperse and homogeneous.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000
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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 41202
Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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