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- EMDB-9015: Apo-EsCas13d -

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Basic information

Entry
Database: EMDB / ID: EMD-9015
TitleApo-EsCas13d
Map dataApo-EsCas13d
Sample
  • Complex: Apo-EsCas13d
    • Protein or peptide: Apo-esCas13d
Biological speciesunidentified bacterium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsZhang C / Lyumkis D
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research InstituteDP5 OD021396 United States
National Institutes of Health/National Human Genome Research InstituteU54GM103368 United States
CitationJournal: Cell / Year: 2018
Title: Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d.
Authors: Cheng Zhang / Silvana Konermann / Nicholas J Brideau / Peter Lotfy / Xuebing Wu / Scott J Novick / Timothy Strutzenberg / Patrick R Griffin / Patrick D Hsu / Dmitry Lyumkis /
Abstract: CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems ...CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
History
DepositionAug 1, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseOct 3, 2018-
UpdateNov 27, 2019-
Current statusNov 27, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9015.png

Downloads & links

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Map

FileDownload / File: emd_9015.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationApo-EsCas13d
Voxel sizeX=Y=Z: 0.79 Å
Density
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-0.15447053 - 0.74998766
Average (Standard dev.)0.0010342313 (±0.029982168)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 202.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.790.790.79
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z202.240202.240202.240
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1540.7500.001

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Supplemental data

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Additional map: Apo-EsCas13d, additional map

Fileemd_9015_additional.map
AnnotationApo-EsCas13d, additional map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Apo-EsCas13d, half map #1

Fileemd_9015_half_map_1.map
AnnotationApo-EsCas13d, half map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Apo-EsCas13d, half map #2

Fileemd_9015_half_map_2.map
AnnotationApo-EsCas13d, half map #2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Apo-EsCas13d

EntireName: Apo-EsCas13d
Components
  • Complex: Apo-EsCas13d
    • Protein or peptide: Apo-esCas13d

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Supramolecule #1: Apo-EsCas13d

SupramoleculeName: Apo-EsCas13d / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: unidentified bacterium (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 110 KDa

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Macromolecule #1: Apo-esCas13d

MacromoleculeName: Apo-esCas13d / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: unidentified bacterium (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGKKIHARDL REQRKTDRTE KFADQNKKRE AERAVPKKDA AVSVKSVSSV SSKKDNVTKS MAKAAGVKSV FAVGNTVYMT SFGRGNDAVL EQKIVDTSHE PLNIDDPAYQ LNVVTMNGYS VTGHRGETVS AVTDNPLRRF NGRKKDEPEQ SVPTDMLCLK PTLEKKFFGK ...String:
MGKKIHARDL REQRKTDRTE KFADQNKKRE AERAVPKKDA AVSVKSVSSV SSKKDNVTKS MAKAAGVKSV FAVGNTVYMT SFGRGNDAVL EQKIVDTSHE PLNIDDPAYQ LNVVTMNGYS VTGHRGETVS AVTDNPLRRF NGRKKDEPEQ SVPTDMLCLK PTLEKKFFGK EFDDNIHIQL IYNILDIEKI LAVYSTNAIY ALNNMSADEN IENSDFFMKR TTDETFDDFE KKKESTNSRE KADFDAFEKF IGNYRLAYFA DAFYVNKKNP KGKAKNVLRE DKELYSVLTL IGKLRHWCVH SEEGRAEFWL YKLDELKDDF KNVLDVVYNR PVEEINNRFI ENNKVNIQIL GSVYKNTDIA ELVRSYYEFL ITKKYKNMGF SIKKLRESML EGKGYADKEY DSVRNKLYQM TDFILYTGYI NEDSDRADDL VNTLRSSLKE DDKTTVYCKE ADYLWKKYRE SIREVADALD GDNIKKLSKS NIEIQEDKLR KCFISYADSV SEFTKLIYLL TRFLSGKEIN DLVTTLINKF DNIRSFLEIM DELGLDRTFT AEYSFFEGST KYLAELVELN SFVKSCSFDI NAKRTMYRDA LDILGIESDK TEEDIEKMID NILQIDANGD KKLKKNNGLR NFIASNVIDS NRFKYLVRYG NPKKIRETAK CKPAVRFVLN EIPDAQIERY YEACCPKNTA LCSANKRREK LADMIAEIKF ENFSDAGNYQ KANVTSRTSE AEIKRKNQAI IRLYLTVMYI MLKNLVNVNA RYVIAFHCVE RDTKLYAESG LEVGNIEKNK TNLTMAVMGV KLENGIIKTE FDKSFAENAA NRYLRNARWY KLILDNLKKS ERAVVNEFRN TVCHLNAIRN ININIKEIKE VENYFALYHY LIQKHLENRF ADKKVERDTG DFISKLEEHK TYCKDFVKAY CTPFGYNLVR YKNLTIDGLF DKNYPGKDDS DEQK

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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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
25.0 mMTris-HClTris
100.0 mMsodium chlorideNaClSodium chloride
5.0 %glycerol
1.0 mMDTT
1.0 mMmagnesium chlorideMgCl2
0.1 %(w/v)Amphipol A8-35
GridPretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 57000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-120 / Number real images: 1158 / Average electron dose: 56.8 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: RELION
Startup modelType of model: OTHER / Details: Apo-EsCas13d
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 15846

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Correlation coefficient

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