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- PDB-6s91: Cryo-EM structure of the Type III-B Cmr-beta bound to cognate tar... -

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Basic information

Entry
Database: PDB / ID: 6s91
TitleCryo-EM structure of the Type III-B Cmr-beta bound to cognate target RNA and AMPPnP, state 2
Components
  • (CRISPR-associated RAMP protein, ...) x 2
  • (CRISPR-associated protein, ...) x 3
  • CRISPR-associated protein Cmrx
  • Cmr1,CRISPR-associated RAMP protein, Cmr1 family
  • Cognate target RNA
  • crRNA (48-MER)
KeywordsANTIVIRAL PROTEIN / CRISPR-Cas / Effector complex / nuclease / cyclic oligo-adenylate synthase
Function / homology
Function and homology information


CRISPR-cas system / defense response to virus / membrane / cytoplasm
Similarity search - Function
CRISPR system CMR subunit Cmr7 1 / CRISPR system CMR subunit Cmr7 1 / : / CRISPR-associated protein, TM1793 / CRISPR-associated protein, TM1791 / CRISPR-associated protein TM1795 / CRISPR-associated protein, Cmr3 / : / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 ...CRISPR system CMR subunit Cmr7 1 / CRISPR system CMR subunit Cmr7 1 / : / CRISPR-associated protein, TM1793 / CRISPR-associated protein, TM1791 / CRISPR-associated protein TM1795 / CRISPR-associated protein, Cmr3 / : / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 / AF1862-like domain superfamily / CRISPR-associated protein Cmr2 / CRISPR-associated protein Cmr2, N-terminal / CRISPR-Cas system, Cmr2 subunit, D1 domain, cysteine cluster / CRISPR-associated protein Cmr2, N-terminal / Cas10/Cmr2, second palm domain / CRISPR type III-associated protein / RAMP superfamily / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / : / : / RNA / RNA (> 10) / CRISPR-associated protein, Cmr3 family / CRISPR-associated protein, Cmr2 family / CRISPR-associated RAMP protein, Cmr6 family / CRISPR-associated RAMP protein, Cmr1 family / CRISPR type III-B/RAMP module-associated protein Cmr5 ...PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / : / : / RNA / RNA (> 10) / CRISPR-associated protein, Cmr3 family / CRISPR-associated protein, Cmr2 family / CRISPR-associated RAMP protein, Cmr6 family / CRISPR-associated RAMP protein, Cmr1 family / CRISPR type III-B/RAMP module-associated protein Cmr5 / CRISPR-associated RAMP protein, Cmr4 family / CRISPR-associated protein Cmrx
Similarity search - Component
Biological speciesSulfolobus islandicus (acidophilic)
Sulfolobus islandicus REY15A (acidophilic)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsSofos, N. / Montoya, G. / Stella, S.
Funding support Denmark, 3items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
Novo Nordisk FoundationNNF0024386 Denmark
Novo Nordisk FoundationNNF17SA0030214 Denmark
Citation
Journal: Mol Cell / Year: 2020
Title: Structures of the Cmr-β Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas.
Authors: Nicholas Sofos / Mingxia Feng / Stefano Stella / Tillmann Pape / Anders Fuglsang / Jinzhong Lin / Qihong Huang / Yingjun Li / Qunxin She / Guillermo Montoya /
Abstract: Cmr-β is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) ...Cmr-β is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-β, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-β and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.
#1: Journal: Biorxiv / Year: 2020
Title: Structures of the Cmr-beta Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas
Authors: Sofos, N. / Feng, M. / Stella, S. / Pape, T. / Fuglsang, A. / Lin, J. / Huang, Q. / Li, Y. / She, Q. / Montoya, G.
History
DepositionJul 11, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 8, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 12, 2020Group: Database references / Category: citation / citation_author
Revision 1.2Sep 16, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Nov 20, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI ..._citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: CRISPR-associated protein, Cmr5 family
B: CRISPR-associated protein, Cmr5 family
C: CRISPR-associated protein, Cmr5 family
D: CRISPR-associated RAMP protein, Cmr4 family
E: CRISPR-associated RAMP protein, Cmr4 family
F: CRISPR-associated RAMP protein, Cmr4 family
G: CRISPR-associated RAMP protein, Cmr4 family
H: CRISPR-associated protein, Cmr3 family
I: CRISPR-associated RAMP protein, Cmr6 family
J: Cmr1,CRISPR-associated RAMP protein, Cmr1 family
K: CRISPR-associated protein, Cmr2 family
U: Cognate target RNA
V: crRNA (48-MER)
L: CRISPR-associated protein Cmrx
M: CRISPR-associated protein Cmrx
N: CRISPR-associated protein Cmrx
O: CRISPR-associated protein Cmrx
P: CRISPR-associated protein Cmrx
Q: CRISPR-associated protein Cmrx
S: CRISPR-associated protein Cmrx
T: CRISPR-associated protein Cmrx
W: CRISPR-associated protein Cmrx
X: CRISPR-associated protein Cmrx
l: CRISPR-associated protein Cmrx
m: CRISPR-associated protein Cmrx
n: CRISPR-associated protein Cmrx
o: CRISPR-associated protein Cmrx
p: CRISPR-associated protein Cmrx
q: CRISPR-associated protein Cmrx
r: CRISPR-associated protein Cmrx
s: CRISPR-associated protein Cmrx
t: CRISPR-associated protein Cmrx
w: CRISPR-associated protein Cmrx
x: CRISPR-associated protein Cmrx
R: CRISPR-associated protein Cmrx
hetero molecules


Theoretical massNumber of molelcules
Total (without water)895,25541
Polymers894,01335
Non-polymers1,2436
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, homology, mass spectrometry, light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area126340 Å2
ΔGint-663 kcal/mol
Surface area272080 Å2
MethodPISA

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Components

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CRISPR-associated protein, ... , 3 types, 5 molecules ABCHK

#1: Protein CRISPR-associated protein, Cmr5 family


Mass: 17961.834 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Source: (natural) Sulfolobus islandicus (strain REY15A) (acidophilic)
Strain: REY15A / References: UniProt: F0NDX5
#3: Protein CRISPR-associated protein, Cmr3 family


Mass: 36008.547 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Sulfolobus islandicus (strain REY15A) (acidophilic)
Strain: REY15A / References: UniProt: F0NDX1
#6: Protein CRISPR-associated protein, Cmr2 family


Mass: 120856.453 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Sulfolobus islandicus (strain REY15A) (acidophilic)
Strain: REY15A / References: UniProt: F0NDX2

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CRISPR-associated RAMP protein, ... , 2 types, 5 molecules DEFGI

#2: Protein
CRISPR-associated RAMP protein, Cmr4 family


Mass: 32322.359 Da / Num. of mol.: 4 / Mutation: D31A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfolobus islandicus (strain REY15A) (acidophilic)
Strain: REY15A / Gene: SiRe_0602 / Plasmid: pAC / Production host: Sulfolobus islandicus REY15A (acidophilic) / References: UniProt: F0NDX6
#4: Protein CRISPR-associated RAMP protein, Cmr6 family


Mass: 33945.914 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal histidine-tag
Source: (gene. exp.) Sulfolobus islandicus (strain REY15A) (acidophilic)
Strain: REY15A / Gene: SiRe_0599 / Plasmid: pAC / Production host: Sulfolobus islandicus REY15A (acidophilic) / References: UniProt: F0NDX3

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Protein , 2 types, 23 molecules JLMNOPQSTWXlmnopqrstwxR

#5: Protein Cmr1,CRISPR-associated RAMP protein, Cmr1 family


Mass: 55388.965 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Sulfolobus islandicus REY15A (acidophilic), (natural) Sulfolobus islandicus (strain REY15A) (acidophilic)
Strain: REY15A / References: UniProt: F0NDX4
#9: Protein ...
CRISPR-associated protein Cmrx


Mass: 19705.607 Da / Num. of mol.: 22 / Source method: isolated from a natural source
Source: (natural) Sulfolobus islandicus (strain REY15A) (acidophilic)
Strain: REY15A / References: UniProt: F0NDX7

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RNA chain , 2 types, 2 molecules UV

#7: RNA chain Cognate target RNA


Mass: 14704.712 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: RNA chain crRNA (48-MER)


Mass: 16409.871 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfolobus islandicus REY15A (acidophilic)
Plasmid: pAC / Production host: Sulfolobus islandicus REY15A (acidophilic) / References: GenBank: 323473489

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Non-polymers , 3 types, 6 molecules

#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#12: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Type III-B Cmr-beta ternary complex, cognate target RNA and AMPPnP boundCOMPLEXCognate target RNA and AMPPnP added to the complex#1-#90MULTIPLE SOURCES
2CRISPR-associated proteins Cmr5, Cmr3, Cmr1, Cmr2, Cmr7COMPLEX#1, #3, #5-#6, #91NATURAL
3CRISPR-associated RAMP proteins Cmr4 and Cmr6, and crRNACOMPLEX#2, #4, #81RECOMBINANT
4Cognate target RNACOMPLEX#71NATURAL
Molecular weightValue: 0.9 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Sulfolobus islandicus REY15A (acidophilic)930945
23Sulfolobus islandicus REY15A (acidophilic)930945
34synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
13Sulfolobus islandicus REY15A (acidophilic)930945
24synthetic construct (others)32630
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
10.15 Msodium chlorideNaCl1
20.01 MMES1
31 mMManganese ChlorideMnCl21
41 mMDTT1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 5 mA (Leica EM ACE200)
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3-4 s blotting before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 96000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1700 nm / Cs: 2.1 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 40 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3912

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
9PHENIXmodel fittingmap_to_model
11PHENIXmodel refinement
12RELION3initial Euler assignmentbeta
13cisTEM1final Euler assignmentbeta
14cisTEM1classificationbeta
15cisTEM13D reconstructionbeta
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 785000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51280 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Cross-correlation coefficient

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