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- EMDB-9014: EsCas13d-crRNA-target RNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-9014
TitleEsCas13d-crRNA-target RNA ternary complex
Map dataEsCas13d-crRNA-target RNA ternary complex
Sample
  • Complex: Ternary complex of EsCas13d(R295A/H300A/R849A/H854A) with crRNA, target RNA and Mg2+
    • Protein or peptide: EsCas13d
    • RNA: crRNA (52-MER)
    • RNA: RNA (27-MER)
  • Ligand: MAGNESIUM ION
KeywordsCRISPR-Cas / RNase / Complex / RNA BINDING PROTEIN-RNA complex
Function / homologyUncharacterized protein
Function and homology information
Biological speciesunidentified bacterium (bacteria) / [Eubacterium] siraeum DSM 15702 (bacteria) / bacterium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsZhang C / Lyumkis D
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)DP5 OD021396 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)U54GM103368 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-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6e9f
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9014.png

Downloads & links

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Map

FileDownload / File: emd_9014.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEsCas13d-crRNA-target RNA ternary complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.79 Å/pix.
x 256 pix.
= 202.24 Å		0.79 Å/pix.
x 256 pix.
= 202.24 Å		0.79 Å/pix.
x 256 pix.
= 202.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.79 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.36141536 - 0.6693507
Average (Standard dev.)0.000044590084 (±0.029999966)
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-0.3610.6690.000

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Supplemental data

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Additional map: 3D FSC map

Fileemd_9014_additional.map
Annotation3D FSC map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EsCas13d-crRNA-target RNA ternary complex, half map #1

Fileemd_9014_half_map_1.map
AnnotationEsCas13d-crRNA-target RNA ternary complex, half map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EsCas13d-crRNA-target RNA ternary complex, half map #2

Fileemd_9014_half_map_2.map
AnnotationEsCas13d-crRNA-target RNA ternary complex, 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 : Ternary complex of EsCas13d(R295A/H300A/R849A/H854A) with crRNA, ...

EntireName: Ternary complex of EsCas13d(R295A/H300A/R849A/H854A) with crRNA, target RNA and Mg2+
Components
  • Complex: Ternary complex of EsCas13d(R295A/H300A/R849A/H854A) with crRNA, target RNA and Mg2+
    • Protein or peptide: EsCas13d
    • RNA: crRNA (52-MER)
    • RNA: RNA (27-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of EsCas13d(R295A/H300A/R849A/H854A) with crRNA, ...

SupramoleculeName: Ternary complex of EsCas13d(R295A/H300A/R849A/H854A) with crRNA, target RNA and Mg2+
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: unidentified bacterium (bacteria)
Molecular weightTheoretical: 135 KDa

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Macromolecule #1: EsCas13d

MacromoleculeName: EsCas13d / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: [Eubacterium] siraeum DSM 15702 (bacteria)
Molecular weightTheoretical: 110.52257 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGKKIHARDL REQRKTDRTE KFADQNKKRE AERAVPKKDA AVSVKSVSSV SSKKDNVTKS MAKAAGVKSV FAVGNTVYMT SFGRGNDAV LEQKIVDTSH EPLNIDDPAY QLNVVTMNGY SVTGHRGETV SAVTDNPLRR FNGRKKDEPE QSVPTDMLCL K PTLEKKFF ...String:
MGKKIHARDL REQRKTDRTE KFADQNKKRE AERAVPKKDA AVSVKSVSSV SSKKDNVTKS MAKAAGVKSV FAVGNTVYMT SFGRGNDAV LEQKIVDTSH EPLNIDDPAY QLNVVTMNGY SVTGHRGETV SAVTDNPLRR FNGRKKDEPE QSVPTDMLCL K PTLEKKFF GKEFDDNIHI QLIYNILDIE KILAVYSTNA IYALNNMSAD ENIENSDFFM KRTTDETFDD FEKKKESTNS RE KADFDAF EKFIGNYRLA YFADAFYVNK KNPKGKAKNV LREDKELYSV LTLIGKLAHW CVASEEGRAE FWLYKLDELK DDF KNVLDV VYNRPVEEIN NRFIENNKVN IQILGSVYKN TDIAELVRSY YEFLITKKYK NMGFSIKKLR ESMLEGKGYA DKEY DSVRN KLYQMTDFIL YTGYINEDSD RADDLVNTLR SSLKEDDKTT VYCKEADYLW KKYRESIREV ADALDGDNIK KLSKS NIEI QEDKLRKCFI SYADSVSEFT KLIYLLTRFL SGKEINDLVT TLINKFDNIR SFLEIMDELG LDRTFTAEYS FFEGST KYL AELVELNSFV KSCSFDINAK RTMYRDALDI LGIESDKTEE DIEKMIDNIL QIDANGDKKL KKNNGLRNFI ASNVIDS NR FKYLVRYGNP KKIRETAKCK PAVRFVLNEI PDAQIERYYE ACCPKNTALC SANKRREKLA DMIAEIKFEN FSDAGNYQ K ANVTSRTSEA EIKRKNQAII RLYLTVMYIM LKNLVNVNAR YVIAFHCVER DTKLYAESGL EVGNIEKNKT NLTMAVMGV KLENGIIKTE FDKSFAENAA NRYLRNARWY KLILDNLKKS ERAVVNEFAN TVCALNAIRN ININIKEIKE VENYFALYHY LIQKHLENR FADKKVERDT GDFISKLEEH KTYCKDFVKA YCTPFGYNLV RYKNLTIDGL FDKNYPGKDD SDEQK

UniProtKB: Uncharacterized protein

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Macromolecule #2: crRNA (52-MER)

MacromoleculeName: crRNA (52-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: bacterium (bacteria)
Molecular weightTheoretical: 16.715051 KDa
SequenceString:
CACCCGUGCA AAAAUGCAGG GGUCUAAAAC GACCUGAAUA UUUCAGAUCA AA

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Macromolecule #3: RNA (27-MER)

MacromoleculeName: RNA (27-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: bacterium (bacteria)
Molecular weightTheoretical: 8.574064 KDa
SequenceString:
ACGUUUUGAU CUGAAAUAUU CAGGUCU

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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-HCl
100.0 mMsodium chlorideNaCl
1.0 mMDTT
5.0 %glycerol
1.0 mMmagnesium chlorideMgCl2
0.1 %(w/v)Amphipol A8-35
GridPretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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: 1-60 / Number real images: 2205 / Average electron dose: 57.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 39000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Details: the ternary complex of EsCas13d with crRNA, target RNA and Mg2+
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 51885
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM

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Atomic model buiding 1

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

PDB-6e9f:
EsCas13d-crRNA-target RNA ternary complex

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