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

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Basic information

Entry
Database: PDB / ID: 6mpu
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 / 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
Specimen sourcePseudomonas aeruginosa (bacteria)
Pseudomonas aeruginosa UCBPP-PA14 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.4 Å resolution
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.
CitationJournal: To be Published
Title: Structure of double-stranded target DNA engaged Csy complex from Pseudomonas aeruginosa (PA-14)
Authors: Rollins, M.F. / Chowdhury, S. / Carter, J. / Golden, S.M. / Miettinen, H.M. / Santiago-Frangos, A. / Faith, D. / Lawrence, M.C. / Wiedenheft, B. / Lander, G.C.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 8, 2018 / Release: Oct 24, 2018

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Structure visualization

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  • Superimposition on EM map
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
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
A: CRISPR-associated protein Csy1


Theoretical massNumber of molelcules
Total (without water)375,91612
Polyers375,91612
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)69010
ΔGint (kcal/M)-201
Surface area (Å2)118820

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Components

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

#1: Protein/peptide CRISPR-associated protein Csy2 / CRISPR type I-f associated protein csy2 (Cas 5f)


Mass: 36244.074 Da / Num. of mol.: 1
Source: (gene. exp.) Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14 / Gene: csy2, PA14_33320 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02MM0
#2: Protein/peptide
CRISPR-associated protein Csy3 / CRISPR type I-f associated protein csy3 (Cas7.1f-7.6f)


Mass: 37579.273 Da / Num. of mol.: 6
Source: (gene. exp.) Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14 / Gene: csy3, csy1-3, PA14_33310 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02MM1
#7: Protein/peptide CRISPR-associated protein Csy1 / CRISPR type I-f associated protein csy1 (Cas 8f)


Mass: 49194.168 Da / Num. of mol.: 1
Source: (gene. exp.) Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14 / Gene: csy1, PA14_33330 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02ML9

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DNA chain , 2 types, 2 molecules NO

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


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


Mass: 10476.714 Da / Num. of mol.: 1
Source: (synth.) Pseudomonas aeruginosa UCBPP-PA14 (bacteria)

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Protein/peptide / RNA chain , 2 types, 2 molecules LM

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


Mass: 21675.781 Da / Num. of mol.: 1
Source: (gene. exp.) Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14 / Gene: cas6f, csy4, PA14_33300 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q02MM2, Acting on Ester Bonds
#4: RNA chain CRISPR RNA (60-MER)


Mass: 19265.404 Da / Num. of mol.: 1
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 / 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: 1, 2, 3, 4, 5, 6, 7 / Source: RECOMBINANT
Molecular weightValue: 0.36 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa (strain 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 type: Quantifoil, UltrAuFoil, R1.2/1.3
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
MicroscopyMicroscope model: 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 / Nominal defocus max: 1500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 79 kelvins / Temperature (min): 77 kelvins
Image recordingAverage exposure time: 14 sec. / Electron dose: 58 e/Å2
Details: Data were acquired using Leginon and collected on K2 summit operating in super-resolution mode.
Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 4 / Number of real images: 3208
Image scansSampling size: 2.5 microns / Width: 7676 / Height: 7420 / Movie frames/image: 56 / Used frames/image: 1-56

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Processing

EM software
IDNameVersionCategoryDetails
2Leginon3.0image acquisitionAutomated data acquisition software
4CTFFIND4CTF correction
7UCSF Chimera1.12model fitting
9RELION2.0initial Euler assignment
10RELION2.1final Euler assignment
13PHENIX1.14model refinement
Image processingDetails: 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.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: 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).
Number of particles selected: 1543677
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 291227 / Algorithm: FOURIER SPACE
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.
Symmetry type: POINT
Atomic model buildingDetails: 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.
Ref protocol: AB INITIO MODEL / Ref space: REAL
Atomic model buildingPDB-ID: 5UZ9

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