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- EMDB-9657: Cryo-EM structure of type III-A Csm-CTR2-dsDNA complex -

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Entry
Database: EMDB / ID: EMD-9657
TitleCryo-EM structure of type III-A Csm-CTR2-dsDNA complex
Map dataCryo-EM structure of Csm-CTR2-dsDNA complex
Sample
  • Complex: type III-A Csm-CTR2-dsDNA complex
    • Complex: type III-A Csm-CTR2-dsDNA complex, Csm1 subunit
      • Protein or peptide: Type III-A CRISPR-associated protein Csm1
    • Protein or peptide: Type III-A CRISPR-associated protein Csm2
    • Protein or peptide: Type III-A CRISPR-associated RAMP protein Csm3
    • Protein or peptide: Type III-A CRISPR-associated RAMP protein Csm4
    • Protein or peptide: Type III-A CRISPR-associated RAMP protein Csm5
    • RNA: crRNA
    • RNA: CTR2
KeywordsCsm complex / Type III-A / CRISPR-Cas system / RNA BINDING PROTEIN
Function / homology
Function and homology information


exonuclease activity / transferase activity / defense response to virus / endonuclease activity / RNA binding / ATP binding
Similarity search - Function
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 ...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 / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / HD domain / HD domain
Similarity search - Domain/homology
CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm5 / CRISPR system Cms protein Csm2 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
Similarity search - Component
Biological speciesStreptococcus thermophilus ND03 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsYou L / Ma J
CitationJournal: Cell / Year: 2019
Title: Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference.
Authors: Lilan You / Jun Ma / Jiuyu Wang / Daria Artamonova / Min Wang / Liang Liu / Hua Xiang / Konstantin Severinov / Xinzheng Zhang / Yanli Wang /
Abstract: Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, ...Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.
History
DepositionSep 21, 2018-
Header (metadata) releaseOct 24, 2018-
Map releaseDec 12, 2018-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.076
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.076
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ifu
  • Surface level: 0.076
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9657.png

Downloads & links

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Map

FileDownload / File: emd_9657.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of Csm-CTR2-dsDNA complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 200 pix.
= 208. Å		1.04 Å/pix.
x 200 pix.
= 208. Å		1.04 Å/pix.
x 200 pix.
= 208. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.076 / Movie #1: 0.076
Minimum - Maximum-0.32534233 - 0.5903137
Average (Standard dev.)-0.0000015661357 (±0.025989076)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 208.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z208.000208.000208.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-0.3250.590-0.000

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Supplemental data

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Additional map: Cryo-EM structure of Csm-CTR2-dsDNA complex, csm1

Fileemd_9657_additional.map
AnnotationCryo-EM structure of Csm-CTR2-dsDNA complex, csm1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : type III-A Csm-CTR2-dsDNA complex

EntireName: type III-A Csm-CTR2-dsDNA complex
Components
  • Complex: type III-A Csm-CTR2-dsDNA complex
    • Complex: type III-A Csm-CTR2-dsDNA complex, Csm1 subunit
      • Protein or peptide: Type III-A CRISPR-associated protein Csm1
    • Protein or peptide: Type III-A CRISPR-associated protein Csm2
    • Protein or peptide: Type III-A CRISPR-associated RAMP protein Csm3
    • Protein or peptide: Type III-A CRISPR-associated RAMP protein Csm4
    • Protein or peptide: Type III-A CRISPR-associated RAMP protein Csm5
    • RNA: crRNA
    • RNA: CTR2

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Supramolecule #1: type III-A Csm-CTR2-dsDNA complex

SupramoleculeName: type III-A Csm-CTR2-dsDNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: type III-A Csm-CTR2-dsDNA complex
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Molecular weightTheoretical: 300 KDa

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Supramolecule #2: type III-A Csm-CTR2-dsDNA complex, Csm1 subunit

SupramoleculeName: type III-A Csm-CTR2-dsDNA complex, Csm1 subunit / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: type III-A Csm-CTR2-dsDNA complex, Csm1 subunit
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)

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Macromolecule #1: Type III-A CRISPR-associated protein Csm1

MacromoleculeName: Type III-A CRISPR-associated protein Csm1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Molecular weightTheoretical: 86.910648 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKKEKIDLFY GALLADIGKV IQRATGERKK HALVGADWFD EIADNQVISD QIRYHMANYQ SDKLGNDHLA YITYIADNIA SGVDRRQSN EESDEDTSAK IWDTYTNQAD IFNVFGAQTD KRYFKPTVLN LKSKPNFASA TYEPFSKGDY AAIATRIKNE L AEFEFNQV ...String:
MKKEKIDLFY GALLADIGKV IQRATGERKK HALVGADWFD EIADNQVISD QIRYHMANYQ SDKLGNDHLA YITYIADNIA SGVDRRQSN EESDEDTSAK IWDTYTNQAD IFNVFGAQTD KRYFKPTVLN LKSKPNFASA TYEPFSKGDY AAIATRIKNE L AEFEFNQV QIDSLLNLFE ATLSFVPSST NTKEIADISL ADHSRLTAAF ALAIYDYLED KGRHNYKEDL FTKVSAFYEE EA FLLASFD LSGIQDFIYN INIATNGAAK QLKARSLYLD FMSEYIADSL LDKLGLNRAN MLYVGGGHAY FVLANTEKTV ETL VQFEKD FNQFLLANFQ TRLYVAFGWG SFAAKDIMSE LNSPESYRQV YQKASRMISK KKISRYDYQT LMLLNRGGKS SERE CEICH SVENLVSYHD QKVCDICRGL YQFSKEIAHD HFIITENEGL PIGPNACLKG VAFEKLSQEA FSRVYVKNDY KAGTV KATH VFVGDYQCDE IYNYAALSKN ENGLGIKRLA VVRLDVDDLG AAFMAGFSQQ GNGQYSTLSR SATFSRSMSL FFKVYI NQF ASDKKLSIIY AGGDDVFAIG SWQDIIAFTV ELRENFIKWT NGKLTLSAGI GLFADKTPIS LMAHQTGELE EAAKGNE KD SISLFSSDYT FKFDRFITNV YDDKLEQIRY FFNHQDERGK NFIYKLIELL RNHDRMNMAR LAYYLTRLEE LTRETDRD K FKTFKNLFYS WYTNKNDKDR KEAELALLLY IYEIRKD

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

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Macromolecule #2: Type III-A CRISPR-associated protein Csm2

MacromoleculeName: Type III-A CRISPR-associated protein Csm2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Molecular weightTheoretical: 14.848145 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MTILTDENYV DIAEKAILKL ERNTRNRKNP DAFFLTTSKL RNLLSLTSTL FDESKVKEYD ALLDRIAYLR VQFVYQAGRE IAVKDLIEK AQILEALKEI KDRETLQRFC RYMEALVAYF KFYGGKD

UniProtKB: CRISPR system Cms protein Csm2

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Macromolecule #3: Type III-A CRISPR-associated RAMP protein Csm3

MacromoleculeName: Type III-A CRISPR-associated RAMP protein Csm3 / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Molecular weightTheoretical: 24.584855 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTFAKIKFSA QIRLETGLHI GGSDAFAAIG AINSPVIKDP ITNLPIIPGS SLKGKMRTLL AKVYNEKVAE KPSDDSDILS RLFGNSKDK RFKMGRLIFR DAFLSNADEL DSLGVRSYTE VKFENTIDRI TAEANPRQIE RAIRNSTFDF ELIYEITDEN E NQVEEDFK ...String:
MTFAKIKFSA QIRLETGLHI GGSDAFAAIG AINSPVIKDP ITNLPIIPGS SLKGKMRTLL AKVYNEKVAE KPSDDSDILS RLFGNSKDK RFKMGRLIFR DAFLSNADEL DSLGVRSYTE VKFENTIDRI TAEANPRQIE RAIRNSTFDF ELIYEITDEN E NQVEEDFK VIRDGLKLLE LDYLGGSGSR GYGKVAFENL KATTVFGNYD VKTLNELLTA EV

UniProtKB: CRISPR system Cms endoribonuclease Csm3

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Macromolecule #4: Type III-A CRISPR-associated RAMP protein Csm4

MacromoleculeName: Type III-A CRISPR-associated RAMP protein Csm4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Molecular weightTheoretical: 33.828984 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTYKLYIMTF QNAHFGSGTL DSSKLTFSAD RIFSALVLEA LKMGKLDAFL AEANQDKFTL TDAFPFQFGP FLPKPIGYPK HDQIDQSVD VKEVRRQAKL SKKLQFLALE NVDDYLNGEL FENEEHAVID TVTKNQPHKD DNLYQVATTR FSNDTSLYVI A NESDLLNE ...String:
MTYKLYIMTF QNAHFGSGTL DSSKLTFSAD RIFSALVLEA LKMGKLDAFL AEANQDKFTL TDAFPFQFGP FLPKPIGYPK HDQIDQSVD VKEVRRQAKL SKKLQFLALE NVDDYLNGEL FENEEHAVID TVTKNQPHKD DNLYQVATTR FSNDTSLYVI A NESDLLNE LMSSLQYSGL GGKRSSGFGR FELDIQNIPL ELSDRLTKNH SDKVMSLTTA LPVDADLEEA MEDGHYLLTK SS GFAFSHA TNENYRKQDL YKFASGSTFS KTFEGQIVDV RPLDFPHAVL NYAKPLFFKL EV

UniProtKB: CRISPR system Cms protein Csm4

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Macromolecule #5: Type III-A CRISPR-associated RAMP protein Csm5

MacromoleculeName: Type III-A CRISPR-associated RAMP protein Csm5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Molecular weightTheoretical: 41.102113 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKNDYRTFKL SLLTLAPIHI GNGEKYTSRE FIYENKKFYF PDMGKFYNKM VEKRLAEKFE AFLIQTRPNA RNNRLISFLN DNRIAERSF GGYSISETGL ESDKNPNSAG AINEVNKFIR DAFGNPYIPG SSLKGAIRTI LMNTTPKWNN ENAVNDFGRF P KENKNLIP ...String:
MKNDYRTFKL SLLTLAPIHI GNGEKYTSRE FIYENKKFYF PDMGKFYNKM VEKRLAEKFE AFLIQTRPNA RNNRLISFLN DNRIAERSF GGYSISETGL ESDKNPNSAG AINEVNKFIR DAFGNPYIPG SSLKGAIRTI LMNTTPKWNN ENAVNDFGRF P KENKNLIP WGPKKGKEYD DLFNAIRVSD SKPFDNKSLI LVQKWDYSAK TNKAKPLPLY RESISPLTKI EFEITTTTDE AG RLIEELG KRAQAFYKDY KAFFLSEFPD DKIQANLQYP IYLGAGSGAW TKTLFKQADG ILQRRYSRMK TKMVKKGVLK LTK APLKTV KIPSGNHSLV KNHESFYEMG KANFMIKEID K

UniProtKB: CRISPR system Cms protein Csm5

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Macromolecule #6: crRNA

MacromoleculeName: crRNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Molecular weightTheoretical: 11.39277 KDa
SequenceString:
ACGGAAACGC UUUCUAGCUC GCUAUAAUUA CCCAUU

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Macromolecule #7: CTR2

MacromoleculeName: CTR2 / type: rna / ID: 7 / Number of copies: 1
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Molecular weightTheoretical: 16.274729 KDa
SequenceString:
GGUAGGAAUG GGUAAUUAUA GCGAGCUAGA AAGCCAAAGG AAGUUUUGUC

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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: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 60 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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

Particle selectionNumber selected: 684150
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 75887
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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