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- PDB-6sh8: Cryo-EM structure of the Type III-B Cmr-beta bound to cognate tar... -

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Basic information

Entry
Database: PDB / ID: 6sh8
TitleCryo-EM structure of the Type III-B Cmr-beta bound to cognate target RNA and AMPPnP, state 2, in the presence of ssDNA
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
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
AF1862-like domain / CRISPR system CMR subunit Cmr7 1 / CRISPR system CMR subunit Cmr7 1 / : / CRISPR-associated protein, TM1793 / CRISPR-associated protein TM1795 / CRISPR-associated protein, TM1791 / : / CRISPR-associated protein, Cmr3 / CRISPR-associated protein (Cas_Cmr3) ...AF1862-like domain / CRISPR system CMR subunit Cmr7 1 / CRISPR system CMR subunit Cmr7 1 / : / CRISPR-associated protein, TM1793 / CRISPR-associated protein TM1795 / CRISPR-associated protein, TM1791 / : / 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 / Peroxidase; domain 1 / Reverse transcriptase/Diguanylate cyclase domain / Orthogonal Bundle / Mainly Alpha
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 REY15A (archaea)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 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
DepositionAug 6, 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 1.3Oct 23, 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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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
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
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
U: Cognate target RNA
V: crRNA
Z: CRISPR-associated protein Cmrx
z: CRISPR-associated protein Cmrx
Y: CRISPR-associated protein Cmrx
y: CRISPR-associated protein Cmrx
hetero molecules


Theoretical massNumber of molelcules
Total (without water)974,07845
Polymers972,83539
Non-polymers1,2436
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, homology, light scattering, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area134650 Å2
ΔGint-706 kcal/mol
Surface area297210 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 REY15A (archaea) / 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 REY15A (archaea) / 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 REY15A (archaea) / 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 REY15A (archaea) / Gene: SiRe_0602 / Production host: Sulfolobus islandicus REY15A (archaea) / 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 for co-purification of the complex.
Source: (gene. exp.) Sulfolobus islandicus REY15A (archaea) / Gene: SiRe_0599 / Production host: Sulfolobus islandicus REY15A (archaea) / References: UniProt: F0NDX3

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Protein , 2 types, 27 molecules JLMNOPQRSTWXlmnopqrstwxZzYy

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


Mass: 55388.965 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: The N-terminal "MEEELL" sequence is not part of the annotated sequence (F0NDX4) due to an annotation mistake.
Source: (natural) Sulfolobus islandicus REY15A (archaea) / References: UniProt: F0NDX4
#7: Protein ...
CRISPR-associated protein Cmrx


Mass: 19705.607 Da / Num. of mol.: 26 / Source method: isolated from a natural source / Source: (natural) Sulfolobus islandicus REY15A (archaea) / References: UniProt: F0NDX7

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RNA chain , 2 types, 2 molecules UV

#8: RNA chain Cognate target RNA


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


Mass: 16409.871 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfolobus islandicus REY15A (archaea) / Plasmid: pAC / Production host: Sulfolobus islandicus REY15A (archaea) / 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 bound, in the presence of ssDNACOMPLEXCognate target RNA, AMPPnP, and ssDNA added to the sample#1-#90MULTIPLE SOURCES
2CRISPR-associated RAMP protein, Cmr4 familyCOMPLEX#21RECOMBINANT
3CRISPR-associated RAMP protein, Cmr6 familyCOMPLEX#41RECOMBINANT
4crRNACOMPLEX#91RECOMBINANT
5CRISPR-associated protein, Cmr5 familyCOMPLEX#11NATURAL
6CRISPR-associated protein, Cmr3 familyCOMPLEX#31NATURAL
7Cmr1,CRISPR-associated RAMP protein, Cmr1 familyCOMPLEX#51NATURAL
8CRISPR-associated protein, Cmr2 familyCOMPLEX#61NATURAL
9CRISPR-associated protein CmrxCOMPLEX#71NATURAL
10Cognate target RNACOMPLEX#81NATURAL
Molecular weightValue: 0.9 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
15Sulfolobus islandicus REY15A (archaea)930945
26Sulfolobus islandicus REY15A (archaea)930945
37Sulfolobus islandicus REY15A (archaea)930945
48Sulfolobus islandicus REY15A (archaea)930945
59Sulfolobus islandicus REY15A (archaea)930945
62Sulfolobus islandicus REY15A (archaea)930945
73Sulfolobus islandicus REY15A (archaea)930945
84Sulfolobus islandicus REY15A (archaea)930945
910synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Sulfolobus islandicus REY15A (archaea)930945
23Sulfolobus islandicus REY15A (archaea)930945
34Sulfolobus islandicus REY15A (archaea)930945
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
10.15 Msodium chlorideNaCl1
20.01 MMES1
31 mMCalcium ChlorideCaCl21
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 5 mA (Leica EM ACE200) / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil
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: 41 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5298

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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: 429000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50409 / 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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