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- EMDB-0516: Small conformation of ssRNA-bound CRISPR_Csm complex -

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Basic information

Entry
Database: EMDB / ID: EMD-0516
TitleSmall conformation of ssRNA-bound CRISPR_Csm complex
Map dataSmall conformation of ssRNA-bound CRISPR_Csm complex
Sample
  • Complex: Small conformation of ssRNA-bound CRISPR_Csm complex
    • Protein or peptide: CRISPR system Cms protein Csm2
    • RNA: crRNA
    • Protein or peptide: CRISPR type III-associated RAMP protein Csm4
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • RNA: target ssRNA
    • Protein or peptide: CRISPR type III-associated RAMP protein Csm3
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsCRISPR / Type III-A / ssRNAase / ssDNase / HYDROLASE / TRANSFERASE-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
Similarity search - Function
: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / 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 ...: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / 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 / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm4 / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm2
Similarity search - Component
Biological speciesStreptococcus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZhang K / Pintilie G
Funding support United States, China, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)P41GM103832 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)U54GM103297 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R01GM079429 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10OD021600 United States
National Natural Science Foundation of China (NSFC)31825008 China
National Natural Science Foundation of China (NSFC)31422014 China
CitationJournal: Cell Res / Year: 2019
Title: Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry.
Authors: Minghui Guo / Kaiming Zhang / Yuwei Zhu / Grigore D Pintilie / Xiaoyu Guan / Shanshan Li / Michael F Schmid / Zhuo Ma / Wah Chiu / Zhiwei Huang /
Abstract: The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading ...The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown. Here we report cryo-EM structures of the Csm-crRNA complex, with or without target ssRNA, at near-atomic resolution. Our cryo-EM maps allow us to build atomic models of the key macromolecular components, including Cas10, Csm2, Csm3, Csm4, crRNA and the invading ssRNA. Our structure resolves unambiguously the stoichiometry and tertiary structures of the Csm protein complex and the interactions between protein components and the crRNA/ssRNA. Interestingly, the new atomic structures of the Csm proteins presented here are similar to those of previously known Csm proteins in other species despite their low sequence similarity. Our combined structural and biochemical data suggest that ssRNA cleavage is preferentially carried out near its 5'-end, that the extent of interactions among the ssRNA, crRNA and the protein components regulates the DNase activity of the Csm complex, and that the 3' flanking sequence of ssRNA activates the Cas10 DNase activity allosterically.
History
DepositionJan 31, 2019-
Header (metadata) releaseFeb 27, 2019-
Map releaseMar 13, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nud
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0516.png

Downloads & links

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Map

FileDownload / File: emd_0516.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSmall conformation of ssRNA-bound CRISPR_Csm complex
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-1.9540308 - 3.347922
Average (Standard dev.)0.00083021907 (±0.0987355)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 407.03998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z407.040407.040407.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-1.9543.3480.001

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Supplemental data

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Sample components

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Entire : Small conformation of ssRNA-bound CRISPR_Csm complex

EntireName: Small conformation of ssRNA-bound CRISPR_Csm complex
Components
  • Complex: Small conformation of ssRNA-bound CRISPR_Csm complex
    • Protein or peptide: CRISPR system Cms protein Csm2
    • RNA: crRNA
    • Protein or peptide: CRISPR type III-associated RAMP protein Csm4
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • RNA: target ssRNA
    • Protein or peptide: CRISPR type III-associated RAMP protein Csm3
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Small conformation of ssRNA-bound CRISPR_Csm complex

SupramoleculeName: Small conformation of ssRNA-bound CRISPR_Csm complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Streptococcus thermophilus (bacteria)

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Macromolecule #1: CRISPR system Cms protein Csm2

MacromoleculeName: CRISPR system Cms protein Csm2 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 14.238391 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MAILTDENYV DKAERAISLL EKDNKGNYLL TTSQIRKLLS LCSSLYDRSK ERKFDELIND VSYLRVQFVY QAGREIAVKD LIEKAQILE ALKEIKDRET LQRFCRYMEA LVAYFKFYGG KD

UniProtKB: CRISPR system Cms protein Csm2

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Macromolecule #3: CRISPR type III-associated RAMP protein Csm4

MacromoleculeName: CRISPR type III-associated RAMP protein Csm4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 33.786949 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MTYKLYIMTF QNAHFGSGTL DSSKLTFSAD RIFSALVLES LKMGKLDAFL AEANQDKFTL TDAFPFQFGP FLPKPIGYPK HDQIDQSVD VKEVRRQAKL SKKLQFLALE NVDDYLNGEL FENEEHAVID TVTKNQPHKD GNLYQVATTR FSNDTSLYVI A NESDLLNE ...String:
MTYKLYIMTF QNAHFGSGTL DSSKLTFSAD RIFSALVLES LKMGKLDAFL AEANQDKFTL TDAFPFQFGP FLPKPIGYPK HDQIDQSVD VKEVRRQAKL SKKLQFLALE NVDDYLNGEL FENEEHAVID TVTKNQPHKD GNLYQVATTR 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 #4: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1...

MacromoleculeName: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 86.930672 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MKKEKIDLFY GALLHDIGKV IQRATGERKK HALVGADWFD EIADNQVISD QIRYHMANYQ SDKLGNDHLA YITYIADNIA SGVDRRQSN EESDEDASAK IWDTYTNQAD IFNVFGAQTD KRYFKPTVLN LKSKPNFASA TYEPFSKGDY AAIATRIKNE L AEFEFNQA ...String:
MKKEKIDLFY GALLHDIGKV IQRATGERKK HALVGADWFD EIADNQVISD QIRYHMANYQ SDKLGNDHLA YITYIADNIA SGVDRRQSN EESDEDASAK IWDTYTNQAD IFNVFGAQTD KRYFKPTVLN LKSKPNFASA TYEPFSKGDY AAIATRIKNE L AEFEFNQA QIDSLLNLFE AILSFVPSST NSKEIADISL AEHSRLTAAF ALAIYDYLED KGRHNYKEDL FTKASAFYEE EA FLLASFD LSGIQDFIYN IATSGAAKQL KARSLYLDFM SEYIADSLLD KLGLNRANLL YVGGGHAYFV LANTEKTVET LVQ FEKDFN QFLLANFQTR LYVAFGWGSF AAKDIMSELN SPESYRQIYQ KASRMISEKK ISRYDYRTLM LLNRGGKSSE RECE ICHSV ENLVSYHDQK VCDICRGLYQ FSKEIAHDHF IITENEGLPI GPNACLKGVA FEKLSQESFS RVYVKNDYKA GTIKA THVF VGDYQCDEIH KYAALSKNED GLGIKRLAVV RLDVDDLGAA FMAGFSRQGN GQYSTLSRSA TFSRSMSLFF KVYINQ FAS DKKLSIIYAG GDDVFAIGSW QDIIAFTVEL RQNFIKWTNG KLTLSAGIGL FADKTPISLM AHQTGELEEA AKGNEKD SI SLFSSDYTFK FDRFITNVYD DKLEQIRYFF NHQDERGKNF IYKLIELLRN YESEEKMNVA RLAYYLTRLE ELTDKDER D KFKQFKKLFF KWYTNNESDR KEAELALLLY VYEIRKD

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

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Macromolecule #6: CRISPR type III-associated RAMP protein Csm3

MacromoleculeName: CRISPR type III-associated RAMP protein Csm3 / type: protein_or_peptide / ID: 6 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 24.556908 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MTFAKIKFSA QIRLETGLHI GGSDAFAAIG AIASPVIKDP ITNIPIIPGS SLKGKMRTLL AKVYNEKVAE KPSDDSDILS RLFGNSKDK RFKMGRLIFR DAFLSNADEL DSLGVRSYTE VKFENTIDRI TAEANPRQIE RAIRNSTFDF ELIYEITDEN E NQVEEDFK ...String:
MTFAKIKFSA QIRLETGLHI GGSDAFAAIG AIASPVIKDP ITNIPIIPGS SLKGKMRTLL AKVYNEKVAE KPSDDSDILS RLFGNSKDK RFKMGRLIFR DAFLSNADEL DSLGVRSYTE VKFENTIDRI TAEANPRQIE RAIRNSTFDF ELIYEITDEN E NQVEEDFK VIRDGLKLLE LDYLGGSGSR GYGKVAFEKL KATTVFGNYD VKTLNELLTA EV

UniProtKB: CRISPR system Cms endoribonuclease Csm3

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 22.935553 KDa
SequenceString:
ACGGAAACUU UCGUAACUGU UUAAUUCUGU UCACUUAUUC CACCGAUAUA AACCUAAUUA CCUCGAGAGG GG

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Macromolecule #5: target ssRNA

MacromoleculeName: target ssRNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 15.980722 KDa
SequenceString:
GGGAAUAAGU GAACAGAAUU AAACAGUUAC GAAAAAAAAA AAGGGUACC

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Macromolecule #7: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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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

Concentration0.4 mg/mL
BufferpH: 7.6
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 7.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 0.65) / Number images used: 50092

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