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- EMDB-45631: Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, ... -

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Basic information

Entry
Database: EMDB / ID: EMD-45631
TitleCas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 1
Map datasharpened map with deepEMhancer
Sample
  • Complex: Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 1
    • Complex: Cas12a
      • Protein or peptide: CRISPR-associated endonuclease Cas12a
    • Complex: gRNA:DNA
      • RNA: guide RNA (25-MER)
      • DNA: Non-target DNA strand
      • DNA: Target DNA strand
KeywordsDNase / complex / ribonucleoprotein / genome editor / HYDROLASE-RNA complex / HYDROLASE / HYDROLASE-RNA-DNA complex
Function / homology
Function and homology information


Bacillus subtilis ribonuclease / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding
Similarity search - Function
: / CRISPR-associated endonuclease Cpf1 REC2 domain / 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: 3.6 Å
AuthorsSoczek KM / Doudna JA
Funding support United States, 6 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Science Foundation (NSF, United States)2334028 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R21HL173710 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)U19NS132303 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19AI171110, U54AI170792, U19AI135990, UH3AI150552, U01AI142817 United States
Department of Energy (DOE, United States)DE-AC02-05CH11231, 2553571, B656358 United States
CitationJournal: Nucleic Acids Res / Year: 2024
Title: CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.
Authors: Katarzyna M Soczek / Joshua C Cofsky / Owen T Tuck / Honglue Shi / Jennifer A Doudna /
Abstract: RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use ...RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.
History
DepositionJul 6, 2024-
Header (metadata) releaseJan 8, 2025-
Map releaseJan 8, 2025-
UpdateJan 8, 2025-
Current statusJan 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45631.png

Downloads & links

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Map

FileDownload / File: emd_45631.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map with deepEMhancer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.12 Å/pix.
x 256 pix.
= 285.44 Å		1.12 Å/pix.
x 256 pix.
= 285.44 Å		1.12 Å/pix.
x 256 pix.
= 285.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.115 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0125
Minimum - Maximum-0.001840936 - 1.9709533
Average (Standard dev.)0.00086492137 (±0.021627959)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 285.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45631_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

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Additional map: unsharpened map from Relion refinement

Fileemd_45631_additional_1.map
Annotationunsharpened map from Relion refinement
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: sharpened map from Relion postprocessing

Fileemd_45631_additional_2.map
Annotationsharpened map from Relion postprocessing
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_45631_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_45631_half_map_2.map
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Sample components

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Entire : Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, ...

EntireName: Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 1
Components
  • Complex: Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 1
    • Complex: Cas12a
      • Protein or peptide: CRISPR-associated endonuclease Cas12a
    • Complex: gRNA:DNA
      • RNA: guide RNA (25-MER)
      • DNA: Non-target DNA strand
      • DNA: Target DNA strand

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Supramolecule #1: Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, ...

SupramoleculeName: Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 185 KDa

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Supramolecule #2: Cas12a

SupramoleculeName: Cas12a / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Acidaminococcus sp. BV3L6 (bacteria)

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Supramolecule #3: gRNA:DNA

SupramoleculeName: gRNA:DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1, #3-#4
Source (natural)Organism: synthetic construct (others) / Synthetically produced: Yes

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Macromolecule #1: guide RNA (25-MER)

MacromoleculeName: guide RNA (25-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.418956 KDa
SequenceString:
UUUAAUUUCU ACUCUUGUAG AUAGCAGAGU AGACAUACGC AG

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Macromolecule #2: CRISPR-associated endonuclease Cas12a

MacromoleculeName: CRISPR-associated endonuclease Cas12a / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: deoxyribonuclease I
Source (natural)Organism: Acidaminococcus sp. BV3L6 (bacteria)
Molecular weightTheoretical: 151.68025 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SNAMTQFEGF TNLYQVSKTL RFELIPQGKT LKHIQEQGFI EEDKARNDHY KELKPIIDRI YKTYADQCLQ LVQLDWENLS AAIDSYRKE KTEETRNALI EEQATYRNAI HDYFIGRTDN LTDAINKRHA EIYKGLFKAE LFNGKVLKQL GTVTTTEHEN A LLRSFDKF ...String:
SNAMTQFEGF TNLYQVSKTL RFELIPQGKT LKHIQEQGFI EEDKARNDHY KELKPIIDRI YKTYADQCLQ LVQLDWENLS AAIDSYRKE KTEETRNALI EEQATYRNAI HDYFIGRTDN LTDAINKRHA EIYKGLFKAE LFNGKVLKQL GTVTTTEHEN A LLRSFDKF TTYFSGFYEN RKNVFSAEDI STAIPHRIVQ DNFPKFKENC HIFTRLITAV PSLREHFENV KKAIGIFVST SI EEVFSFP FYNQLLTQTQ IDLYNQLLGG ISREAGTEKI KGLNEVLNLA IQKNDETAHI IASLPHRFIP LFKQILSDRN TLS FILEEF KSDEEVIQSF CKYKTLLRNE NVLETAEALF NELNSIDLTH IFISHKKLET ISSALCDHWD TLRNALYERR ISEL TGKIT KSAKEKVQRS LKHEDINLQE IISAAGKELS EAFKQKTSEI LSHAHAALDQ PLPTTLKKQE EKEILKSQLD SLLGL YHLL DWFAVDESNE VDPEFSARLT GIKLEMEPSL SFYNKARNYA TKKPYSVEKF KLNFQMPTLA SGWDVNKEKC NGAILF VKN GLYYLGIMPK QKGRYKALSF EPTEKTSEGF DKMYYDYFPD AAKMIPKCST QLKAVTAHFQ THTTPILLSN NFIEPLE IT KEIYDLNNPE KEPKKFQTAY AKKTGDQKGY REALCKWIDF TRDFLSKYTK TTSIDLSSLR PSSQYKDLGE YYAELNPL L YHISFQRIAE KEIMDAVETG KLYLFQIYNK DFAKGHHGKP NLHTLYWTGL FSPENLAKTS IKLNGQAELF YRPKSRMKR MAHRLGEKML NKKLKDQKTP IPDTLYQELY DYVNHRLSHD LSDEARALLP NVITKEVSHE IIKDRRFTSD KFFFHVPITL NYQAANSPS KFNQRVNAYL KEHPETPIIG IDRGERNLIY ITVIDSTGKI LEQRSLNTIQ QFDYQKKLDN REKERVAARQ A WSVVGTIK DLKQGYLSQV IHEIVDLMIH YQAVVVLENL NFGFKSKRTG IAEKAVYQQF EKMLIDKLNC LVLKDYPAEK VG GVLNPYQ LTDQFTSFAK MGTQSGFLFY VPAPYTSKID PLTGFVDPFV WKTIKNHESR KHFLEGFDFL HYDVKTGDFI LHF KMNRNL SFQRGLPGFM PAWDIVFEKN ETQFDAKGTP FIAGKRIVPV IENHRFTGRY RDLYPANELI ALLEEKGIVF RDGS NILPK LLENDDSHAI DTMVALIRSV LQMRNSNAAT GEDYINSPVR DLNGVCFDSR FQNPEWPMDA DANGAYHIAL KGQLL LNHL KESKDLKLQN GISNQDWLAY IQELRN

UniProtKB: CRISPR-associated endonuclease Cas12a

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Macromolecule #3: Non-target DNA strand

MacromoleculeName: Non-target DNA strand / type: dna / ID: 3
Details: cytidine in position 7 is covalently modified with XL1, forming N4-cystamine-2'-deoxycytidine; the modified base is forming a disulfide bond with cysteine 551 in the protein
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.567206 KDa
SequenceString:
(DG)(DT)(DA)(DG)(DT)(DG)(2YR)(DT)(DT)(DT) (DG)(DT)(DC)(DG)(DT)(DC)(DT)(DC)(DA) (DT)(DC)(DT)(DG)(DT)(DA)(DT)(DG)(DC)(DG) (DT)(DC)

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Macromolecule #4: Target DNA strand

MacromoleculeName: Target DNA strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.557205 KDa
SequenceString:
(DG)(DA)(DC)(DG)(DC)(DA)(DT)(DA)(DC)(DA) (DG)(DA)(DT)(DG)(DA)(DG)(DA)(DC)(DG)(DA) (DC)(DA)(DA)(DA)(DG)(DC)(DA)(DC)(DT) (DA)(DC)

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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

BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMTris-Cl
200.0 mMpotassium chlorideKCl
0.1 mMdithiothreitol
5.0 mMmagnesium chlorideMgCl2
0.25 %glycerol
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: ab initio reconstruction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5-beta) / Number images used: 20031
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5-beta)
Final 3D classificationSoftware - Name: RELION (ver. 5-beta)
FSC plot (resolution estimation)

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