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- EMDB-40441: WT CRISPR-Cas12a with a 5bp R-loop -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-40441
TitleWT CRISPR-Cas12a with a 5bp R-loop
Map data5bp R-loop unsharpened map
Sample
  • Complex: WT AsCas12a incubated with 8bp-complementary target DNA.
    • Protein or peptide: CRISPR-associated endonuclease Cas12a
    • RNA: RNA (27-MER)
    • DNA: DNA (32-MER)
    • DNA: DNA (32-MER)
KeywordsCRISPR / R-loop / endonuclease / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


Bacillus subtilis ribonuclease / Bacillus subtilis ribonuclease activity / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding
Similarity search - Function
CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas12a
Similarity search - Component
Biological speciesAcidaminococcus sp. BV3L6 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsStrohkendl I / Taylor DW
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: To Be Published
Title: WT CRISPR-Cas12a with a 5bp R-loop
Authors: Strohkendl I / Taylor DW
History
DepositionApr 11, 2023-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40441.png

Downloads & links

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Map

FileDownload / File: emd_40441.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation5bp R-loop unsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 266.56 Å		0.83 Å/pix.
x 320 pix.
= 266.56 Å		0.83 Å/pix.
x 320 pix.
= 266.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.833 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.275
Minimum - Maximum-0.29757813 - 0.888422
Average (Standard dev.)0.0013373017 (±0.03264052)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: 5bp R-loop half map A

Fileemd_40441_half_map_1.map
Annotation5bp R-loop half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 5bp R-loop half map B

Fileemd_40441_half_map_2.map
Annotation5bp R-loop half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : WT AsCas12a incubated with 8bp-complementary target DNA.

EntireName: WT AsCas12a incubated with 8bp-complementary target DNA.
Components
  • Complex: WT AsCas12a incubated with 8bp-complementary target DNA.
    • Protein or peptide: CRISPR-associated endonuclease Cas12a
    • RNA: RNA (27-MER)
    • DNA: DNA (32-MER)
    • DNA: DNA (32-MER)

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Supramolecule #1: WT AsCas12a incubated with 8bp-complementary target DNA.

SupramoleculeName: WT AsCas12a incubated with 8bp-complementary target DNA.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Acidaminococcus sp. BV3L6 (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas12a

MacromoleculeName: CRISPR-associated endonuclease Cas12a / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: deoxyribonuclease I
Source (natural)Organism: Acidaminococcus sp. BV3L6 (bacteria)
Molecular weightTheoretical: 151.418953 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTQFEGFTNL YQVSKTLRFE LIPQGKTLKH IQEQGFIEED KARNDHYKEL KPIIDRIYKT YADQCLQLVQ LDWENLSAAI DSYRKEKTE ETRNALIEEQ ATYRNAIHDY FIGRTDNLTD AINKRHAEIY KGLFKAELFN GKVLKQLGTV TTTEHENALL R SFDKFTTY ...String:
MTQFEGFTNL YQVSKTLRFE LIPQGKTLKH IQEQGFIEED KARNDHYKEL KPIIDRIYKT YADQCLQLVQ LDWENLSAAI DSYRKEKTE ETRNALIEEQ ATYRNAIHDY FIGRTDNLTD AINKRHAEIY KGLFKAELFN GKVLKQLGTV TTTEHENALL R SFDKFTTY FSGFYENRKN VFSAEDISTA IPHRIVQDNF PKFKENCHIF TRLITAVPSL REHFENVKKA IGIFVSTSIE EV FSFPFYN QLLTQTQIDL YNQLLGGISR EAGTEKIKGL NEVLNLAIQK NDETAHIIAS LPHRFIPLFK QILSDRNTLS FIL EEFKSD EEVIQSFCKY KTLLRNENVL ETAEALFNEL NSIDLTHIFI SHKKLETISS ALCDHWDTLR NALYERRISE LTGK ITKSA KEKVQRSLKH EDINLQEIIS AAGKELSEAF KQKTSEILSH AHAALDQPLP TTLKKQEEKE ILKSQLDSLL GLYHL LDWF AVDESNEVDP EFSARLTGIK LEMEPSLSFY NKARNYATKK PYSVEKFKLN FQMPTLASGW DVNKEKNNGA ILFVKN GLY YLGIMPKQKG RYKALSFEPT EKTSEGFDKM YYDYFPDAAK MIPKCSTQLK AVTAHFQTHT TPILLSNNFI EPLEITK EI YDLNNPEKEP KKFQTAYAKK TGDQKGYREA LCKWIDFTRD FLSKYTKTTS IDLSSLRPSS QYKDLGEYYA ELNPLLYH I SFQRIAEKEI MDAVETGKLY LFQIYNKDFA KGHHGKPNLH TLYWTGLFSP ENLAKTSIKL NGQAELFYRP KSRMKRMAH RLGEKMLNKK LKDQKTPIPD TLYQELYDYV NHRLSHDLSD EARALLPNVI TKEVSHEIIK DRRFTSDKFF FHVPITLNYQ AANSPSKFN QRVNAYLKEH PETPIIGIDR GERNLIYITV IDSTGKILEQ RSLNTIQQFD YQKKLDNREK ERVAARQAWS V VGTIKDLK QGYLSQVIHE IVDLMIHYQA VVVLENLNFG FKSKRTGIAE KAVYQQFEKM LIDKLNCLVL KDYPAEKVGG VL NPYQLTD QFTSFAKMGT QSGFLFYVPA PYTSKIDPLT GFVDPFVWKT IKNHESRKHF LEGFDFLHYD VKTGDFILHF KMN RNLSFQ RGLPGFMPAW DIVFEKNETQ FDAKGTPFIA GKRIVPVIEN HRFTGRYRDL YPANELIALL EEKGIVFRDG SNIL PKLLE NDDSHAIDTM VALIRSVLQM RNSNAATGED YINSPVRDLN GVCFDSRFQN PEWPMDADAN GAYHIALKGQ LLLNH LKES KDLKLQNGIS NQDWLAYIQE LRN

UniProtKB: CRISPR-associated endonuclease Cas12a

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Macromolecule #2: RNA (27-MER)

MacromoleculeName: RNA (27-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.598088 KDa
SequenceString:
AAUUUCUACU CUUGUAGAUG UGAUAAG

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Macromolecule #3: DNA (32-MER)

MacromoleculeName: DNA (32-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.882405 KDa
SequenceString:
(DA)(DC)(DC)(DG)(DT)(DA)(DA)(DG)(DG)(DT) (DC)(DT)(DT)(DA)(DT)(DC)(DA)(DC)(DT)(DA) (DA)(DA)(DA)(DG)(DA)(DT)(DC)(DG)(DG) (DA)(DA)(DG)

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Macromolecule #4: DNA (32-MER)

MacromoleculeName: DNA (32-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.797312 KDa
SequenceString:
(DC)(DT)(DT)(DC)(DC)(DG)(DA)(DT)(DC)(DT) (DT)(DT)(DT)(DA)(DG)(DT)(DG)(DA)(DT)(DA) (DA)(DG)(DA)(DC)(DC)(DT)(DT)(DA)(DC) (DG)(DG)(DT)

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 60595
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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