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- EMDB-9653: Cryo-EM structure of type III-A Csm-CTR1 complex, AMPPNP bound -

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Basic information

Entry
Database: EMDB / ID: EMD-9653
TitleCryo-EM structure of type III-A Csm-CTR1 complex, AMPPNP bound
Map dataCryo-EM structure of Csm-CTR1, AMPPNP bound
Sample
  • Complex: Csm-CTR1 complex, AMPPNP bound
    • 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: CTR1
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
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: 3.2 Å
AuthorsYou L / Ma J / Wang J / Zhang X / Wang Y
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 20, 2018-
Header (metadata) releaseOct 24, 2018-
Map releaseDec 12, 2018-
UpdateJan 23, 2019-
Current statusJan 23, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ifk
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9653.png

Downloads & links

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Map

FileDownload / File: emd_9653.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of Csm-CTR1, AMPPNP bound
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: 0.065 / Movie #1: 0.065
Minimum - Maximum-0.21827108 - 0.3935017
Average (Standard dev.)0.0009108987 (±0.018539451)
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-0.2180.3940.001

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Supplemental data

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Sample components

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Entire : Csm-CTR1 complex, AMPPNP bound

EntireName: Csm-CTR1 complex, AMPPNP bound
Components
  • Complex: Csm-CTR1 complex, AMPPNP bound
    • 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: CTR1
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Csm-CTR1 complex, AMPPNP bound

SupramoleculeName: Csm-CTR1 complex, AMPPNP bound / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7 / Details: type III-A CRISPR-Cas interference complex
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)
Molecular weightTheoretical: 290 KDa

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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: 86.976734 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKKEKIDLFY GALLHNIGKV IQRATGERKK HALVGADWFD EIADNQVISD QIRYHMANYQ SDKLGNDHLA YITYIADNIA SGVDRRQSN EESDEDTSAK IWDTYTNQAD IFNVFGAQTD KRYFKPTVLN LKSKPNFASA TYEPFSKGDY AAIATRIKNE L AEFEFNQV ...String:
MKKEKIDLFY GALLHNIGKV 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

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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

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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

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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

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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

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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: 10.780438 KDa
SequenceString:
ACGGAAACGC UUUCUAGCUC GCUAUAAUUA CCCA

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Macromolecule #7: CTR1

MacromoleculeName: CTR1 / type: rna / ID: 7 / Number of copies: 1
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Molecular weightTheoretical: 13.701244 KDa
SequenceString:
GGUAGGAAUG GGUAAUUAUA GCGAGCUAGA AAGCCAAAGG UC

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Macromolecule #8: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 8 / 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 #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 10 / Number of copies: 1 / 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: 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

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 61538
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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