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- EMDB-42378: Structure of PsCas9 in complex with gRNA and DNA in product state -

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Basic information

Entry
Database: EMDB / ID: EMD-42378
TitleStructure of PsCas9 in complex with gRNA and DNA in product state
Map data
Sample
  • Complex: PsCas9 in with gRNA and dsDNA, product state
    • Protein or peptide: Cas9
    • RNA: RNA (121-MER)
    • DNA: DNA (80-MER)
    • DNA: DNA (80-MER)
  • Ligand: MAGNESIUM ION
KeywordsCas9 / CRISPR / IMMUNE SYSTEM
Biological speciesParasutterella secunda (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsBravo JPK / Taylor DW
Funding support United States, 2 items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: To Be Published
Title: Structure-guided engineering of PsCas9 yields a high fidelity and activity enzyme for in vivo gene editing
Authors: Bravo JPK / Taylor DW
History
DepositionOct 17, 2023-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42378.png

Downloads & links

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Map

FileDownload / File: emd_42378.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 420 pix.
= 340.2 Å		0.81 Å/pix.
x 420 pix.
= 340.2 Å		0.81 Å/pix.
x 420 pix.
= 340.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.81 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0867
Minimum - Maximum-0.2823708 - 0.6996153
Average (Standard dev.)0.00010056169 (±0.013924834)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 340.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_42378_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_42378_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PsCas9 in with gRNA and dsDNA, product state

EntireName: PsCas9 in with gRNA and dsDNA, product state
Components
  • Complex: PsCas9 in with gRNA and dsDNA, product state
    • Protein or peptide: Cas9
    • RNA: RNA (121-MER)
    • DNA: DNA (80-MER)
    • DNA: DNA (80-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: PsCas9 in with gRNA and dsDNA, product state

SupramoleculeName: PsCas9 in with gRNA and dsDNA, product state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Parasutterella secunda (bacteria)
Molecular weightTheoretical: 211.54 KDa

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Macromolecule #1: Cas9

MacromoleculeName: Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Parasutterella secunda (bacteria)
Molecular weightTheoretical: 168.224266 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAASMKRTAD GSEFESPKKK RKVTVKSTLA VDMGGRYTGI FSYTTDSGFP KAKEARAYVL NMPDNDALTY SMAARTQTRH RIRSQQRFV LARRLTYILI EGKLKRKLSP REKEAISSLL RRRGYSRLES ELDLSVLQGV ESGFFKCFLP NFDEDENLLT Q WTSLTDGY ...String:
GAASMKRTAD GSEFESPKKK RKVTVKSTLA VDMGGRYTGI FSYTTDSGFP KAKEARAYVL NMPDNDALTY SMAARTQTRH RIRSQQRFV LARRLTYILI EGKLKRKLSP REKEAISSLL RRRGYSRLES ELDLSVLQGV ESGFFKCFLP NFDEDENLLT Q WTSLTDGY LQNNSDSRRQ IQIFLESSKD SKEFLTVVKS QHQDTKEYKN ALKVMRDDAE SMIEQSMFGH KHRRLYLEAI AQ DIPRDSR LKPIIEAFSG VEKFHHFIGN LSNLQLRALR WYFNDPSMKN NVFDKERLKS VLVRAYQFFH YPKDLTQQRA EVL NAYEGA TDILETLQTL NPELTIPPYE DQNNRRPPLD QTLWLSPRLL DQRYGDTWEI WVQNLLRSPL SKGIDENLDT ILIT TDRKA RLLERQSGRL IHYTSQKLYH SYVLQRLLDR TVENDAYLLK TLVSSNRGNS NEIHQAQERL TRDLGSQHIK KFLDF VRQY YDEVDKAKRG LWFIVEKPLM ERADIHPPMK NDSVILRLVG NILCVSDLVD LSFWTRKVKG QSTVRSLCTA IEKTRK EYG NSFNYLYQRA LYLQSKGKKL SAEDKDFIKL QSNVLLVSDV IAEALDIKEE QKKKFANPFS LAQLYNIIET EKSGFIS TT LAAVDENAWR NNLQGKARCV QLCADTVRPF DGALRNILDR QAYEIAKLKA EELLSTELKN QTIDLVVLLE SNQFAFSA S LAEVKKSANT AAIRQKVAKA QKRQQDRWLS KDERIKSASR GLCPYTGKNL GDKGEVDHII PRSLSMNYMG SILNSEANL IYCSQEGNQL KLNGRKKLSD LADNYLKVVF GTADRGTICK YIEKSVSELT DAKIVQFELL DRSQQDAVRH ALFLEDFSEA RRRIIRLLG KINTARVNGT QAWFAKSFIT KLRELTKEWC ANNQITLAFD LYRLDAQTVS QDYRKKFALI NKDWAKPDDK K QPIASHAI DAFCVFAAAK DKRNIANVLG VFDEVAEEQN LKTIAQLMPS EVNLISPKRK SILDKNEVGS RALMKEGIFA EH FLPILVR GDDCRIGFDW SESGSVKVKD ADKLFGVLDG LLKQSQKRSV NGFETYTVDR IKAFELLHDV FIRPCSQKML EQA EVLEKL HYITQNISVT SVYDAVNRQF KCREEILKDK DFDIKVDLGN RFGSAKGKIT LPAKREWEKL VNRSELKNLI KDKL SDKGS EKTPDGETLI YDIFRSIPVQ KLSHKATRRV WSLPKIPSIS SGVRIKRKDS NGNDIYQLYM LNDTKCKGFV VNEKG VIDW SSDLVADLYK QPTLTILNGR YLKADQYVRM DQWYEVDCGR DDVIVKMCPG TSGRRYIEIT QSKKQFEDWT GYISGS FWN YPVTIKLSSQ QIANFVKNSQ MPLLGKPRSG QITVITLGNT LKYWYCVESK NSMMNEAYQK AYLVHFNQSG GSASDYK DD DDKKRTADGS EFEPKKKRKV

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Macromolecule #2: RNA (121-MER)

MacromoleculeName: RNA (121-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 41.934762 KDa
SequenceString:
AUGUCACCUC CAAUGACUAG GGGUUUCAGU UAUUCGUGAA AACGAAUGAA GUCACUCUUA AAGUGAGCUG AAAUCACUAA AAAUUAAGA UUGAACCCGG CUACUGACUC UGUCAUCCGG GUUUACUUAU UU

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Macromolecule #3: DNA (80-MER)

MacromoleculeName: DNA (80-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 24.8169 KDa
SequenceString: (DT)(DA)(DA)(DG)(DC)(DA)(DG)(DG)(DC)(DC) (DA)(DA)(DT)(DG)(DG)(DG)(DG)(DA)(DG)(DG) (DA)(DC)(DA)(DT)(DC)(DG)(DA)(DT)(DG) (DT)(DC)(DA)(DC)(DC)(DT)(DC)(DC)(DA)(DA) (DT) (DG)(DA)(DC)(DT)(DA)(DG) ...String:
(DT)(DA)(DA)(DG)(DC)(DA)(DG)(DG)(DC)(DC) (DA)(DA)(DT)(DG)(DG)(DG)(DG)(DA)(DG)(DG) (DA)(DC)(DA)(DT)(DC)(DG)(DA)(DT)(DG) (DT)(DC)(DA)(DC)(DC)(DT)(DC)(DC)(DA)(DA) (DT) (DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG) (DT)(DG)(DG)(DG)(DC)(DA)(DA)(DC)(DC)(DA) (DC)(DA) (DA)(DA)(DC)(DC)(DC)(DA)(DC) (DG)(DA)(DG)(DG)(DG)(DC)(DA)(DG)(DA)(DG) (DT)(DG)(DC)

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Macromolecule #4: DNA (80-MER)

MacromoleculeName: DNA (80-MER) / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 24.5306 KDa
SequenceString: (DG)(DC)(DA)(DC)(DT)(DC)(DT)(DG)(DC)(DC) (DC)(DT)(DC)(DG)(DT)(DG)(DG)(DG)(DT)(DT) (DT)(DG)(DT)(DG)(DG)(DT)(DT)(DG)(DC) (DC)(DC)(DA)(DC)(DC)(DC)(DT)(DA)(DG)(DT) (DC) (DA)(DT)(DT)(DG)(DG)(DA) ...String:
(DG)(DC)(DA)(DC)(DT)(DC)(DT)(DG)(DC)(DC) (DC)(DT)(DC)(DG)(DT)(DG)(DG)(DG)(DT)(DT) (DT)(DG)(DT)(DG)(DG)(DT)(DT)(DG)(DC) (DC)(DC)(DA)(DC)(DC)(DC)(DT)(DA)(DG)(DT) (DC) (DA)(DT)(DT)(DG)(DG)(DA)(DG)(DG) (DT)(DG)(DA)(DC)(DA)(DT)(DC)(DG)(DA)(DT) (DG)(DT) (DC)(DC)(DT)(DC)(DC)(DC)(DC) (DA)(DT)(DT)(DG)(DG)(DC)(DC)(DT)(DG)(DC) (DT)(DT)(DA)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 7 / 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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN 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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77059
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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