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

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Basic information

Entry
Database: PDB / ID: 6shb
TitleCryo-EM structure of the Type III-B Cmr-beta bound to cognate target RNA and AMPPnP, state 1, 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 (43-MER)
  • crRNA (49-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-associated RAMP Cmr4 / AF1862-like domain superfamily ...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 / 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 REY15A (acidophilic)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.07 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

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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
I: CRISPR-associated RAMP protein, Cmr6 family
J: Cmr1,CRISPR-associated RAMP protein, Cmr1 family
U: Cognate target RNA (43-MER)
V: crRNA (49-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
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
K: CRISPR-associated protein, Cmr2 family
Z: CRISPR-associated protein Cmrx
z: CRISPR-associated protein Cmrx
H: CRISPR-associated protein, Cmr3 family
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
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, homology, light scattering, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area132840 Å2
ΔGint-748 kcal/mol
Surface area303220 Å2
MethodPISA

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Components

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

#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 (acidophilic)
References: UniProt: F0NDX5
#8: 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 (acidophilic)
References: UniProt: F0NDX2
#9: 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 (acidophilic)
References: UniProt: F0NDX1

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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
Source method: isolated from a genetically manipulated source
Details: Asp31 mutated to Ala
Source: (gene. exp.) Sulfolobus islandicus REY15A (acidophilic)
Gene: SiRe_0602 / Production host: Sulfolobus islandicus REY15A (acidophilic) / References: UniProt: F0NDX6
#3: 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 REY15A (acidophilic)
Gene: SiRe_0599 / Production host: Sulfolobus islandicus REY15A (acidophilic) / References: UniProt: F0NDX3

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Protein , 2 types, 27 molecules JLMNOPQRSTWXlmnopqrstwxZzYy

#4: 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)
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 (acidophilic)
References: UniProt: F0NDX7

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RNA chain , 2 types, 2 molecules UV

#5: RNA chain Cognate target RNA (43-MER)


Mass: 14704.712 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: RNA chain crRNA (49-MER)


Mass: 16409.871 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfolobus islandicus REY15A (acidophilic)
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

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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#31RECOMBINANT
4crRNA (49-MER)COMPLEX#61RECOMBINANT
5CRISPR-associated protein, Cmr5 familyCOMPLEX#11NATURAL
6Cmr1,CRISPR-associated RAMP protein, Cmr1 familyCOMPLEX#41NATURAL
7Cognate target RNA (43-MER)COMPLEX#51NATURAL
8CRISPR-associated protein CmrxCOMPLEX#71NATURAL
9CRISPR-associated protein, Cmr2 familyCOMPLEX#81NATURAL
10CRISPR-associated protein, Cmr3 familyCOMPLEX#91NATURAL
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
34Sulfolobus islandicus REY15A (acidophilic)930945
45Sulfolobus islandicus REY15A (acidophilic)930945
56Sulfolobus islandicus REY15A (acidophilic)930945
67synthetic construct (others)32630
78Sulfolobus islandicus REY15A (acidophilic)930945
89Sulfolobus islandicus REY15A (acidophilic)930945
910Sulfolobus islandicus REY15A (acidophilic)930945
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Sulfolobus islandicus REY15A (acidophilic)930945
23Sulfolobus islandicus REY15A (acidophilic)930945
34Sulfolobus islandicus REY15A (acidophilic)930945
47Sulfolobus islandicus REY15A (acidophilic)930945
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
10.15 Msodium chlorideNaClSodium chloride1
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 FIELDBright-field microscopy / 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
1RELION3particle selectionbeta
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: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65610 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Cross-correlation coefficient

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