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- EMDB-18694: Quaternary complex of ReChb - crRNA - target dsDNA - collateral dsDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-18694
TitleQuaternary complex of ReChb - crRNA - target dsDNA - collateral dsDNA
Map data
Sample
  • Complex: Quaternary complex of ReChb - crRNA - target dsDNA - collateral dsDNA
    • Protein or peptide: ReChb
    • RNA: crRNA
    • DNA: non target DNA
    • DNA: target DNA
  • Ligand: MAGNESIUM ION
KeywordsCas / BIOSYNTHETIC PROTEIN / cryo-EM / ancestral sequence reconstruction / biotechnology / DNA / RNA
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsLopez-Alonso JP / Ubarretxena-Belandia I / Tascon I
Funding support1 items
OrganizationGrant numberCountry
Other private
CitationJournal: Nature Biotechnology / Year: 2024
Title: A resurrected ancestor of Cas12a expands target access and substrate 2 recognition for nucleic-acid editing and detection
Authors: Jabalera Y / Tascon I / Samperio S / Lopez-Alonso JP / Gonzalez-Lopez M / Aransay AM / Abascal-Palacios G / Beisel CL / Ubarretxena-Belandia I / Jimenez-Perez R
History
DepositionOct 19, 2023-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18694.png

Downloads & links

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Map

FileDownload / File: emd_18694.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 320 pix.
= 263.616 Å		0.82 Å/pix.
x 320 pix.
= 263.616 Å		0.82 Å/pix.
x 320 pix.
= 263.616 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8238 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-1.0241269 - 1.5673323
Average (Standard dev.)0.0005876191 (±0.028130855)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 263.616 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18694_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18694_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18694_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Quaternary complex of ReChb - crRNA - target dsDNA - collateral dsDNA

EntireName: Quaternary complex of ReChb - crRNA - target dsDNA - collateral dsDNA
Components
  • Complex: Quaternary complex of ReChb - crRNA - target dsDNA - collateral dsDNA
    • Protein or peptide: ReChb
    • RNA: crRNA
    • DNA: non target DNA
    • DNA: target DNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Quaternary complex of ReChb - crRNA - target dsDNA - collateral dsDNA

SupramoleculeName: Quaternary complex of ReChb - crRNA - target dsDNA - collateral dsDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: ReChb

MacromoleculeName: ReChb / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 148.954109 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPKMFSNFTN QYPLSKTLRF ELKPVGKTLE HIEKKGLLEQ DEKRAEDYKK VKKIIDEYHK DFIEEALNNV KLNGEGLEEY YELYFKKNK DDKDKKKKEF EKIQDNLRKQ IVEAFKNHEK YKNLFKKELI KEDLPNWLKN SEDTGEEDKE TVEKFKNFTT Y FTGFHENR ...String:
GPKMFSNFTN QYPLSKTLRF ELKPVGKTLE HIEKKGLLEQ DEKRAEDYKK VKKIIDEYHK DFIEEALNNV KLNGEGLEEY YELYFKKNK DDKDKKKKEF EKIQDNLRKQ IVEAFKNHEK YKNLFKKELI KEDLPNWLKN SEDTGEEDKE TVEKFKNFTT Y FTGFHENR KNMYSDEEKS TAIAYRLIHE NLPKFLDNMK VFEKIKEKHP EAEQLEKTLK DLGEEEVLGG NNVEDIFSLD YF NHTLTQS GIDIYNTIIG GKTEEDGNKK IQGLNEYINL YRQKNNEKNR KLPKLKPLYK QILSDRESLS FIPEAFENDE ELL EAIEEF YENLNFSNNN EATNVLEKLK ELLSNLADYD LNKIYIRNDT SLTDISQKIF GDWSVIKDAL NAHYDQTYPK KKKK KSKEK LEEKREKWLK KQKYFSIAEL QEALDSYCKE SDESKEQKEN SIADYFKTLA QTKNETDKKT DLIENIKSKY QYPND KKLA QDKEFKDVEK IKAFLDSIMN LQHFVKPLHL VKGGSAGAEM EKDEAFYSEF EALYEELSQV IPLYNKVRNY LTQKPY STE KIKLNFENST LLDGWDVNKE TDNTSVLLRK DGLYYLGIMN KKHNKVFENI PESNENDKCY EKMDYKLLPG ANKMLPK VF FSNKNIDYFN PSAEILEIYE NGTHKKSGDN FNLDDCHKLI DFFKESINKH EDWKKFGFKF SPTSSYEDIS GFYREVEQ Q GYKISFKNIS ESYIDELVDE GKLYLFQIYN KDFSPYSKGK PNLHTLYWKA LFDEENLKDV VYKLNGEAEV FYRKASINE TIVHKANEPI KNKNPLNPKK QSTFEYDIIK DRRYTVDKFQ FHVPITMNFK AEGNSNINDE VNEFLKGNAP DVNIIGIDRG ERHLLYLTL IDQKGKIVEQ DSLNTITNEH NETDYHALLD DKEKERDKAR KSWGTIENIK ELKEGYLSQV VHKIAKLMVE H NAIVVMED LNFGFKRGRF KVEKQVYQKF EKMLIDKLNY LVDKDKEPNE PGGLLNAYQL TNKFESFQKM GKQSGFLFYV PA WNTSKID PTTGFVNLFH PRYENVEKAK EFFNKFDSIR YNSEKDYFEF AFDYNNFTEK AEGTKWTVCT YGERIKTYRN ADK NNQWDS KEVNVTEEFK NLFDEYNIDY KNGNDLKEAI LSQDDADFFK SLLHLLRLTL QMRNSITGTE IDYIISPVAN ENGE FFDSR KADESLPKDA DANGAYHIAR KGLWVLEQIK QTDDLKKVNL AISNKEWLEF VQERKN

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.696498 KDa
SequenceString:
GAAUUUCUAC UGUUGUAGAU UCCUAAGGCG UUACCCCAAU

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Macromolecule #3: non target DNA

MacromoleculeName: non target DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.983707 KDa
SequenceString:
(DC)(DC)(DC)(DT)(DC)(DC)(DC)(DA)(DT)(DT) (DG)(DG)(DG)(DG)(DT)(DA)(DA)(DC)(DG)(DC) (DC)(DT)(DT)(DA)(DG)(DG)(DA)(DT)(DA) (DA)(DA)(DT)(DA)(DT)(DG)(DC)(DT)(DA)(DG)

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Macromolecule #4: target DNA

MacromoleculeName: target DNA / type: dna / ID: 4 / Details: Ctagcatatttatcctaaggcgttaccccaatgggaggg / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.023731 KDa
SequenceString:
(DC)(DT)(DA)(DG)(DC)(DA)(DT)(DA)(DT)(DT) (DT)(DA)(DT)(DC)(DC)(DT)(DA)(DA)(DG)(DG) (DC)(DG)(DT)(DT)(DA)(DC)(DC)(DC)(DC) (DA)(DA)(DT)(DG)(DG)(DG)(DA)(DG)(DG)(DG)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 6 / 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

Concentration2.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
50.0 mMTris
2.0 mMMagnesium chlorideMgCl2
150.0 mMSodium chlorideNaCl
0.05 %CHAPS
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: LEICA EM GP / Details: 2s blotting.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 34123 / Average exposure time: 2.12 sec. / Average electron dose: 50.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
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

Particle selectionNumber selected: 1526906
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio reconstruction
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.4.0) / Number images used: 300509
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.0)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 4.3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: other / Details: ModelAngelo
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8qwf:
ReChb - crRNA - target dsDNA complex in the presence of collateral dsDNA

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