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- EMDB-18691: Apo ReChb -

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Basic information

Entry
Database: EMDB / ID: EMD-18691
TitleApo ReChb
Map data
Sample
  • Complex: Apo ReChb
    • Protein or peptide: ReChb
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsCas / BIOSYNTHETIC PROTEIN / cryo-EM / ancestral sequence reconstruction / biotechnology
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsLopez-Alonso JP / Ubarretxena-Belandia I / Tascon I
Funding support1 items
OrganizationGrant numberCountry
Other government
CitationJournal: Nat Biotechnol / Year: 2024
Title: A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection.
Authors: Ylenia Jabalera / Igor Tascón / Sara Samperio / Jorge P López-Alonso / Monika Gonzalez-Lopez / Ana M Aransay / Guillermo Abascal-Palacios / Chase L Beisel / Iban Ubarretxena-Belandia / Raul Perez-Jimenez /
Abstract: The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs ...The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.
History
DepositionOct 19, 2023-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18691.png

Downloads & links

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Map

FileDownload / File: emd_18691.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: 0.123
Minimum - Maximum-0.95902807 - 1.3185118
Average (Standard dev.)0.000008781542 (±0.021368243)
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_18691_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18691_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_18691_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 : Apo ReChb

EntireName: Apo ReChb
Components
  • Complex: Apo ReChb
    • Protein or peptide: ReChb
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Apo ReChb

SupramoleculeName: Apo ReChb / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
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 BL21(DE3) (bacteria)
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: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 4 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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
150.0 mMsodium chlorideNaCl
2.0 mMmagnesium chlorideMgCl2
0.05 %CHAPS
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: LEICA EM GP

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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: 14874 / Average exposure time: 2.1 sec. / Average electron dose: 49.8 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: 1967948
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: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.4.0) / Number images used: 254316
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. 3.4.0)
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-8qwd:
Apo ReChb

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