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- PDB-6ne0: Structure of double-stranded target DNA engaged Csy complex from ... -

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Basic information

Entry
Database: PDB / ID: 6ne0
TitleStructure of double-stranded target DNA engaged Csy complex from Pseudomonas aeruginosa (PA-14)
Components
  • (CRISPR-associated protein ...) x 3
  • CRISPR RNA (60-MER)
  • CRISPR target DNA (44-MER)
  • CRISPR-associated endonuclease Cas6/Csy4
  • Non-complementary R-loop DNA strand
KeywordsIMMUNE SYSTEM/RNA / type I-F CRISPR RNA-guided surveillance complex / viral protein mimic / Csy-complex / IMMUNE SYSTEM-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding
Similarity search - Function
CRISPR-associated protein Csy1 / CRISPR-associated protein (Cas_Csy1) / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3)
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated protein Csy1 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy3 / CRISPR-associated endonuclease Cas6/Csy4
Similarity search - Component
Biological speciesPseudomonas aeruginosa UCBPP-PA14 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsChowdhury, S. / Rollins, M.F. / Carter, J. / Golden, S.M. / Miettinen, H.M. / Santiago-Frangos, A. / Faith, D. / Lawrence, M.C. / Wiedenheft, B. / Lander, G.C.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2EB020402, P20GM103500, P30GM110732, R01GM110270, R01GM108888,R21AI130670 United States
National Science Foundation (NSF, United States)EPS-110134 United States
CitationJournal: Mol Cell / Year: 2019
Title: Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry.
Authors: MaryClare F Rollins / Saikat Chowdhury / Joshua Carter / Sarah M Golden / Heini M Miettinen / Andrew Santiago-Frangos / Dominick Faith / C Martin Lawrence / Gabriel C Lander / Blake Wiedenheft /
Abstract: Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present ...Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.
History
DepositionDec 15, 2018Deposition site: RCSB / Processing site: RCSB
SupersessionDec 26, 2018ID: 6MPU
Revision 1.0Dec 26, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 30, 2019Group: Data collection / Structure summary / Category: entity / Item: _entity.pdbx_description
Revision 1.2Mar 20, 2019Group: Data collection / Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.3Mar 27, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.4Apr 17, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.5Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Dec 25, 2019Group: Database references / Category: database_2
Revision 1.7Mar 13, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Movie
  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-9191
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated protein Csy1
B: CRISPR-associated protein Csy2
C: CRISPR-associated protein Csy3
D: CRISPR-associated protein Csy3
E: CRISPR-associated protein Csy3
F: CRISPR-associated protein Csy3
G: CRISPR-associated protein Csy3
H: CRISPR-associated protein Csy3
L: CRISPR-associated endonuclease Cas6/Csy4
M: CRISPR RNA (60-MER)
N: CRISPR target DNA (44-MER)
O: Non-complementary R-loop DNA strand


Theoretical massNumber of molelcules
Total (without water)375,91612
Polymers375,91612
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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CRISPR-associated protein ... , 3 types, 8 molecules ABCDEFGH

#1: Protein CRISPR-associated protein Csy1 / CRISPR type I-f associated protein csy1 (Cas 8f)


Mass: 49194.168 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Gaps within the structure correspond to regions that could not be modeled due to missing map density.
Source: (gene. exp.) Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Strain: UCBPP-PA14 / Gene: csy1, PA14_33330 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02ML9
#2: Protein CRISPR-associated protein Csy2 / CRISPR type I-f associated protein csy2 (Cas 5f)


Mass: 36244.074 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Gaps within the structure correspond to regions that could not be modeled due to missing map density.
Source: (gene. exp.) Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Strain: UCBPP-PA14 / Gene: csy2, PA14_33320 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02MM0
#3: Protein
CRISPR-associated protein Csy3 / CRISPR type I-f associated protein csy3 (Cas7.1f-7.6f)


Mass: 37579.273 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Details: Gaps within the structure correspond to regions that could not be modeled due to missing map density.
Source: (gene. exp.) Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Strain: UCBPP-PA14 / Gene: csy3, csy1-3, PA14_33310 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02MM1

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DNA chain , 2 types, 2 molecules NO

#6: DNA chain CRISPR target DNA (44-MER)


Mass: 13584.703 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
#7: DNA chain Non-complementary R-loop DNA strand


Mass: 10476.714 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Gaps within the structure correspond to regions that could not be modeled due to missing map density.
Source: (synth.) Pseudomonas aeruginosa UCBPP-PA14 (bacteria)

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Protein / RNA chain , 2 types, 2 molecules LM

#4: Protein CRISPR-associated endonuclease Cas6/Csy4 / CRISPR type I-f associated protein csy4 (Cas6f)


Mass: 21675.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Strain: UCBPP-PA14 / Gene: cas6f, csy4, PA14_33300 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q02MM2, Hydrolases; Acting on ester bonds
#5: RNA chain CRISPR RNA (60-MER)


Mass: 19265.404 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Gaps within the structure correspond to regions that could not be modeled due to missing map density.
Source: (synth.) Pseudomonas aeruginosa UCBPP-PA14 (bacteria)

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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

ComponentName: DOUBLE-STRANDED TARGET DNA ENGAGED CSY COMPLEX FROM PSEUDOMONAS AERUGINOSA (PA-14)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.36 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMPotassium ChlorideKCl1
31 mMTCEPC9H15O6P1
42 %(v/v)GlycerolC3H8O31
50.05 %(v/v)Lauryl Maltose Neopentyl GlycolC47H88O221
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: Freezing was carried out in a cold room at 4 degrees C and relative humidity of 98%. 5 ul sample was applied to plasma cleaned grid and manually blotted with Whatman 1 filter paper for 5-7 ...Details: Freezing was carried out in a cold room at 4 degrees C and relative humidity of 98%. 5 ul sample was applied to plasma cleaned grid and manually blotted with Whatman 1 filter paper for 5-7 sec before plunge freezing.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Details: OBJECTIVE ASTIGMATISM WAS CORRECTED AT 36000X MAGNIFICATION USING THON RINGS VISUALIZED WITH A K2 CAMERA.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Image recordingElectron dose: 0.58 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3208

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Processing

SoftwareName: PHENIX / Version: dev_3304: / Classification: refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
Image processingDetails: Super-resolution movie frames were Fourier-binned 2X2 times to a pixel size of 1.15 Angstrom/pixel prior to dose-weighted frame alignment using MotionCorr2.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1543677
Details: Particles were initially extracted binned by 2 with a box size of 144 pixels (2.3 Angstrom/pixel). The final processing was carried out with an unbinned particle stack with a box size of 288 ...Details: Particles were initially extracted binned by 2 with a box size of 144 pixels (2.3 Angstrom/pixel). The final processing was carried out with an unbinned particle stack with a box size of 288 pixels (1.15 Angstrom/pixel).
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 291227 / Algorithm: FOURIER SPACE
Details: The final map was reconstructed using several focused maps from different sub-regions of the complex. These were initially aligned to each other and then stitched using the vop maximum ...Details: The final map was reconstructed using several focused maps from different sub-regions of the complex. These were initially aligned to each other and then stitched using the vop maximum function in UCSF chimera. The resolution of the final composite map is 3.2 angstrom.
Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: THE ATOMIC MODELS FOR CAS5F, CAS8F, CAS6F, AND CAS7F FROM THE CSY ACR COMPLEX (PDB ID 5UZ9) WERE USED AS INITIAL TEMPLATE MODELS FOR MODEL BUILDING. THESE WERE INDIVIDUALLY RIGID BODY-FITTED ...Details: THE ATOMIC MODELS FOR CAS5F, CAS8F, CAS6F, AND CAS7F FROM THE CSY ACR COMPLEX (PDB ID 5UZ9) WERE USED AS INITIAL TEMPLATE MODELS FOR MODEL BUILDING. THESE WERE INDIVIDUALLY RIGID BODY-FITTED INTO THE RECONSTRUCTED MAPS USING THE FIT MAP FUNCTION IN UCSF CHIMERA, AND RESIDUE REGISTERS AND BACKBONE GEOMETRIES WERE FIXED IN COOT. MODELS FOR THE CRRNA AND DNA STRANDS WERE ALSO MANUALLY BUILT INTO THE MAP USING COOT.
Atomic model buildingPDB-ID: 5UZ9

5uz9


Accession code: 5UZ9 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.4 Å

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