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- EMDB-40445: WT CRISPR-Cas12a with a 16bp R-loop and nontarget strand in the R... -

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Basic information

Entry
Database: EMDB / ID: EMD-40445
TitleWT CRISPR-Cas12a with a 16bp R-loop and nontarget strand in the RuvC active site.
Map data16bp R-loop unsharpened map
Sample
  • Complex: WT AsCas12a incubated with 16bp-complementary target DNA.
    • Protein or peptide: CRISPR-associated endonuclease Cas12a
    • RNA: RNA (36-MER)
    • DNA: DNA (31-MER)
    • DNA: DNA (30-MER)
KeywordsCRISPR / R-loop / endonuclease / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA 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: 3.3 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: Mol Cell / Year: 2024
Title: Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.
Authors: Isabel Strohkendl / Aakash Saha / Catherine Moy / Alexander-Hoi Nguyen / Mohd Ahsan / Rick Russell / Giulia Palermo / David W Taylor /
Abstract: The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables ...The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.
History
DepositionApr 11, 2023-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40445.png

Downloads & links

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Map

FileDownload / File: emd_40445.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation16bp 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.
= 266.624 Å		0.83 Å/pix.
x 320 pix.
= 266.624 Å		0.83 Å/pix.
x 320 pix.
= 266.624 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.19
Minimum - Maximum-0.31229243 - 0.7749559
Average (Standard dev.)0.00007263048 (±0.025127651)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.624 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: 16bp R-loop half map A

Fileemd_40445_half_map_1.map
Annotation16bp R-loop half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 16bp R-loop half map B

Fileemd_40445_half_map_2.map
Annotation16bp 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 16bp-complementary target DNA.

EntireName: WT AsCas12a incubated with 16bp-complementary target DNA.
Components
  • Complex: WT AsCas12a incubated with 16bp-complementary target DNA.
    • Protein or peptide: CRISPR-associated endonuclease Cas12a
    • RNA: RNA (36-MER)
    • DNA: DNA (31-MER)
    • DNA: DNA (30-MER)

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Supramolecule #1: WT AsCas12a incubated with 16bp-complementary target DNA.

SupramoleculeName: WT AsCas12a incubated with 16bp-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: 151.705234 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GAASMTQFEG FTNLYQVSKT LRFELIPQGK TLKHIQEQGF IEEDKARNDH YKELKPIIDR IYKTYADQCL QLVQLDWENL SAAIDSYRK EKTEETRNAL IEEQATYRNA IHDYFIGRTD NLTDAINKRH AEIYKGLFKA ELFNGKVLKQ LGTVTTTEHE N ALLRSFDK ...String:
GAASMTQFEG FTNLYQVSKT LRFELIPQGK TLKHIQEQGF IEEDKARNDH YKELKPIIDR IYKTYADQCL QLVQLDWENL SAAIDSYRK EKTEETRNAL IEEQATYRNA IHDYFIGRTD NLTDAINKRH AEIYKGLFKA ELFNGKVLKQ LGTVTTTEHE N ALLRSFDK FTTYFSGFYE NRKNVFSAED ISTAIPHRIV QDNFPKFKEN CHIFTRLITA VPSLREHFEN VKKAIGIFVS TS IEEVFSF PFYNQLLTQT QIDLYNQLLG GISREAGTEK IKGLNEVLNL AIQKNDETAH IIASLPHRFI PLFKQILSDR NTL SFILEE FKSDEEVIQS FCKYKTLLRN ENVLETAEAL FNELNSIDLT HIFISHKKLE TISSALCDHW DTLRNALYER RISE LTGKI TKSAKEKVQR SLKHEDINLQ EIISAAGKEL SEAFKQKTSE ILSHAHAALD QPLPTTLKKQ EEKEILKSQL DSLLG LYHL LDWFAVDESN EVDPEFSARL TGIKLEMEPS LSFYNKARNY ATKKPYSVEK FKLNFQMPTL ASGWDVNKEK NNGAIL FVK NGLYYLGIMP KQKGRYKALS FEPTEKTSEG FDKMYYDYFP DAAKMIPKCS TQLKAVTAHF QTHTTPILLS NNFIEPL EI TKEIYDLNNP EKEPKKFQTA YAKKTGDQKG YREALCKWID FTRDFLSKYT KTTSIDLSSL RPSSQYKDLG EYYAELNP L LYHISFQRIA EKEIMDAVET GKLYLFQIYN KDFAKGHHGK PNLHTLYWTG LFSPENLAKT SIKLNGQAEL FYRPKSRMK RMAHRLGEKM LNKKLKDQKT PIPDTLYQEL YDYVNHRLSH DLSDEARALL PNVITKEVSH EIIKDRRFTS DKFFFHVPIT LNYQAANSP SKFNQRVNAY LKEHPETPII GIDRGERNLI YITVIDSTGK ILEQRSLNTI QQFDYQKKLD NREKERVAAR Q AWSVVGTI KDLKQGYLSQ VIHEIVDLMI HYQAVVVLEN LNFGFKSKRT GIAEKAVYQQ FEKMLIDKLN CLVLKDYPAE KV GGVLNPY QLTDQFTSFA KMGTQSGFLF YVPAPYTSKI DPLTGFVDPF VWKTIKNHES RKHFLEGFDF LHYDVKTGDF ILH FKMNRN LSFQRGLPGF MPAWDIVFEK NETQFDAKGT PFIAGKRIVP VIENHRFTGR YRDLYPANEL IALLEEKGIV FRDG SNILP KLLENDDSHA IDTMVALIRS VLQMRNSNAA TGEDYINSPV RDLNGVCFDS RFQNPEWPMD ADANGAYHIA LKGQL LLNH LKESKDLKLQ NGISNQDWLA YIQELRN

UniProtKB: CRISPR-associated endonuclease Cas12a

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Macromolecule #2: RNA (36-MER)

MacromoleculeName: RNA (36-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.351995 KDa
SequenceString:
UUUUUAAUUU CUACUCUUGU AGAUGUGAUA AGUGGAAUGC CAUGUGGA

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Macromolecule #3: DNA (31-MER)

MacromoleculeName: DNA (31-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 17.211082 KDa
SequenceString: (DA)(DG)(DC)(DA)(DC)(DA)(DG)(DT)(DA)(DG) (DC)(DT)(DA)(DC)(DT)(DC)(DC)(DA)(DG)(DT) (DA)(DC)(DG)(DC)(DA)(DT)(DT)(DC)(DC) (DA)(DC)(DT)(DT)(DA)(DT)(DC)(DA)(DC)(DT) (DA) (DA)(DA)(DA)(DG)(DA)(DT) ...String:
(DA)(DG)(DC)(DA)(DC)(DA)(DG)(DT)(DA)(DG) (DC)(DT)(DA)(DC)(DT)(DC)(DC)(DA)(DG)(DT) (DA)(DC)(DG)(DC)(DA)(DT)(DT)(DC)(DC) (DA)(DC)(DT)(DT)(DA)(DT)(DC)(DA)(DC)(DT) (DA) (DA)(DA)(DA)(DG)(DA)(DT)(DC)(DG) (DG)(DA)(DA)(DG)(DA)(DG)(DC)(DG)

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Macromolecule #4: DNA (30-MER)

MacromoleculeName: DNA (30-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 17.299061 KDa
SequenceString: (DC)(DG)(DC)(DT)(DC)(DT)(DT)(DC)(DC)(DG) (DA)(DT)(DC)(DT)(DT)(DT)(DT)(DA)(DG)(DT) (DG)(DA)(DT)(DA)(DA)(DG)(DT)(DG)(DG) (DA)(DA)(DT)(DG)(DC)(DG)(DT)(DA)(DC)(DT) (DG) (DG)(DA)(DG)(DT)(DA)(DG) ...String:
(DC)(DG)(DC)(DT)(DC)(DT)(DT)(DC)(DC)(DG) (DA)(DT)(DC)(DT)(DT)(DT)(DT)(DA)(DG)(DT) (DG)(DA)(DT)(DA)(DA)(DG)(DT)(DG)(DG) (DA)(DA)(DT)(DG)(DC)(DG)(DT)(DA)(DC)(DT) (DG) (DG)(DA)(DG)(DT)(DA)(DG)(DC)(DT) (DA)(DC)(DT)(DG)(DT)(DG)(DC)(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: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 97787
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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