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- EMDB-0442: Type III-A CRISPR Effector Subcomplex from Staphylococcus epiderm... -

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Basic information

Entry
Database: EMDB / ID: EMD-0442
TitleType III-A CRISPR Effector Subcomplex from Staphylococcus epidermidis RP62a
Map dataType III-A CRISPR Effector Subcomplex from Staphylococcus epidermidis RP62a, primary map
Sample
  • Complex: Type III-A CRISPR Effector Subcomplex from Staphylococcus epidermidis RP62a
Biological speciesStaphylococcus epidermidis RP62A (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsDorsey BW / Mondragon A
CitationJournal: Nucleic Acids Res / Year: 2019
Title: Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM.
Authors: Bryan W Dorsey / Lei Huang / Alfonso Mondragón /
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas ...Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.
History
DepositionDec 13, 2018-
Header (metadata) releaseJan 16, 2019-
Map releaseFeb 13, 2019-
UpdateMay 1, 2019-
Current statusMay 1, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0442.png

Downloads & links

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Map

FileDownload / File: emd_0442.map.gz / Format: CCP4 / Size: 55.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationType III-A CRISPR Effector Subcomplex from Staphylococcus epidermidis RP62a, primary map
Voxel sizeX=Y=Z: 1.24 Å
Density
Contour LevelBy AUTHOR: 0.0185 / Movie #1: 0.01
Minimum - Maximum-0.027626785 - 0.0711628
Average (Standard dev.)0.00000088811134 (±0.0019536428)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions244244244
Spacing244244244
CellA=B=C: 302.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.241.241.24
M x/y/z244244244
origin x/y/z0.0000.0000.000
length x/y/z302.560302.560302.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS244244244
D min/max/mean-0.0280.0710.000

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Supplemental data

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Sample components

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Entire : Type III-A CRISPR Effector Subcomplex from Staphylococcus epiderm...

EntireName: Type III-A CRISPR Effector Subcomplex from Staphylococcus epidermidis RP62a
Components
  • Complex: Type III-A CRISPR Effector Subcomplex from Staphylococcus epidermidis RP62a

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Supramolecule #1: Type III-A CRISPR Effector Subcomplex from Staphylococcus epiderm...

SupramoleculeName: Type III-A CRISPR Effector Subcomplex from Staphylococcus epidermidis RP62a
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
Molecular weightExperimental: 255 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.20 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
500.0 mMNaClSodium chlorideSodium chloride
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Details: Glow discharged with 15mA.
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Blot for 4 seconds with +5 force before plunging..

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Electron microscopy

MicroscopeJEOL 3200FS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 40323 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 30000
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
TemperatureMin: 100.0 K / Max: 100.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-40 / Number grids imaged: 7 / Number real images: 1037 / Average exposure time: 8.0 sec. / Average electron dose: 37.5 e/Å2

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Image processing

Particle selectionNumber selected: 277413
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 107620
FSC plot (resolution estimation)

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