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- EMDB-9191: Structure of double-stranded target DNA engaged Csy complex from ... -

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Basic information

Entry
Database: EMDB / ID: 9191
TitleStructure of double-stranded target DNA engaged Csy complex from Pseudomonas aeruginosa (PA-14)
Map dataComposite sharpened map of the complex generated from multiple focused maps of different sub-regions of the complex. A B-factor of -35 was used for sharpening the map.
SampleDouble-stranded target DNA engaged Csy Complex from Pseudomonas aeruginosa (PA-14)
  • (CRISPR-associated protein ...) x 3
  • CRISPR-associated endonuclease Cas6/Csy4
  • (nucleic-acidNucleic acid) x 3
Function / homologyCRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated protein Csy1 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy1) / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein (Cas_Csy3) / CRISPR-associated protein (Cas_Csy4) / maintenance of CRISPR repeat elements / endonuclease activity ...CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated protein Csy1 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy1) / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein (Cas_Csy3) / CRISPR-associated protein (Cas_Csy4) / maintenance of CRISPR repeat elements / endonuclease activity / Acting on Ester Bonds / RNA binding / CRISPR-associated protein Csy1 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy3 / CRISPR-associated endonuclease Cas6/Csy4
Function and homology information
SourcePseudomonas aeruginosa (strain UCBPP-PA14) (bacteria) / Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.4 Å resolution
AuthorsChowdhury S / Rollins MF / Carter J / Golden SM / Miettinen HM / Santiago-Frangos A / Faith D / Lawrence MC / Wiedenheft B / Lander GC
CitationJournal: To be Published
Title: Structure of double-stranded target DNA engaged Csy complex from Pseudomonas aeruginosa (PA-14)
Authors: Rollins MF / Chowdhury S / Carter J / Golden SM / Miettinen HM / Santiago-Frangos A / Faith D / Lawrence MC / Wiedenheft B / Lander GC
Validation ReportPDB-ID: 6mpu

SummaryFull reportAbout validation report
DateDeposition: Oct 8, 2018 / Header (metadata) release: Oct 24, 2018 / Map release: Oct 24, 2018 / Last update: Oct 24, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0595
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0595
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6mpu
  • Surface level: 0.0595
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_9191.map.gz (map file in CCP4 format, 16385 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
160 pix
1.15 Å/pix.
= 184. Å
160 pix
1.15 Å/pix.
= 184. Å
160 pix
1.15 Å/pix.
= 184. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density
Contour Level:0.0595 (by author), 0.0595 (movie #1):
Minimum - Maximum-0.121736825 - 0.27672002
Average (Standard dev.)0.0024644134 (0.016295806)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions160160160
Origin0.00.00.0
Limit159.0159.0159.0
Spacing160160160
CellA=B=C: 184.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z184.000184.000184.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.1220.2770.002

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Supplemental data

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Mask #1

Fileemd_9191_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire Double-stranded target DNA engaged Csy Complex from Pseudomonas a...

EntireName: Double-stranded target DNA engaged Csy Complex from Pseudomonas aeruginosa (PA-14)
Number of components: 8

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Component #1: protein, Double-stranded target DNA engaged Csy Complex from Pseu...

ProteinName: Double-stranded target DNA engaged Csy Complex from Pseudomonas aeruginosa (PA-14)
Recombinant expression: No
MassTheoretical: 360 kDa
SourceSpecies: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #2: protein, CRISPR-associated protein Csy2

ProteinName: CRISPR-associated protein Csy2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 36.244074 kDa
SourceSpecies: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, CRISPR-associated protein Csy3

ProteinName: CRISPR-associated protein Csy3 / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 37.579273 kDa
SourceSpecies: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, CRISPR-associated endonuclease Cas6/Csy4

ProteinName: CRISPR-associated endonuclease Cas6/Csy4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 21.675781 kDa
SourceSpecies: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: nucleic-acid, CRISPR RNA (60-MER)

Nucleic-acidName: CRISPR RNA (60-MER) / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CUAAGAAAUU CACGGCGGGC UUGAUGUCCG CGUCUACCUG GUUCACUGCC GUGUAGGCAG
MassTheoretical: 19.265404 kDa
SourceSpecies: Pseudomonas aeruginosa UCBPP-PA14 (bacteria)

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Component #6: nucleic-acid, CRISPR target DNA (44-MER)

Nucleic-acidName: CRISPR target DNA (44-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DA)(DG)(DG)(DT)(DA)(DG)(DA)(DC)(DG) (DC)(DG)(DG)(DA)(DC)(DA)(DT)(DC)(DA)(DA) (DG)(DC)(DC)(DC)(DG)(DC)(DC)(DG)(DT)(DG) (DA)(DA)(DG)(DG)(DT)(DG)(DC)(DA)(DG)(DC) (DT)(DT)(DC)(DT)
MassTheoretical: 13.584703 kDa
SourceSpecies: Pseudomonas aeruginosa UCBPP-PA14 (bacteria)

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Component #7: nucleic-acid, Non-complementary R-loop DNA strand

Nucleic-acidName: Non-complementary R-loop DNA strand / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DG)(DA)(DA)(DG)(DC)(DT)(DG)(DC)(DA) (DC)(DC)(DT)(DT)(DC)(DA)(DC)(DG)(DG)(DC) (DG)(DG)(DG)(DC)(DT)(DT)(DG)(DA)(DT)(DG) (DT)(DC)(DC)(DG)
MassTheoretical: 10.476714 kDa
SourceSpecies: Pseudomonas aeruginosa UCBPP-PA14 (bacteria)

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Component #8: protein, CRISPR-associated protein Csy1

ProteinName: CRISPR-associated protein Csy1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 49.194168 kDa
SourceSpecies: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 2 mg/ml / pH: 7.5
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 sec before plunge freezing..

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: 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 / Electron dose: 58 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 36000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 600.0 - 1500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: K ( 77.0 - 79.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3208 / Sampling size: 2.5 microns
Details: Data were acquired using Leginon and collected on K2 summit operating in super-resolution mode.

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 291227
Details: Super-resolution movie frames were Fourier-binned 2 x 2 times to a pixel size of 1.15 Angstrom/pixel prior to dose-weighted frame alignment using MotionCorr2.
3D reconstructionAlgorithm: FOURIER SPACE / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF
Details: The final map was reconstructed using several focused maps from different subregions 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.
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement 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 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.
Input PDB model: 5UZ9
Output model

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