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- PDB-6wc0: Crystal structure of AceCas9 bound with guide RNA and DNA with 5'... -

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Basic information

Entry
Database: PDB / ID: 6wc0
TitleCrystal structure of AceCas9 bound with guide RNA and DNA with 5'-NNNTC-3' PAM
Components
  • CRISPR-associated endonuclease, Csn1 family
  • DNA (30-MER)
  • DNA (5'-D(*AP*TP*AP*CP*TP*TP*GP*GP*CP*G)-3')
  • sgRNA (95-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / DNA endonuclease / CRISPR-Cas9 / HNH / RuvC / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


endonuclease activity / defense response to virus / DNA binding / RNA binding / zinc ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, beta-hairpin domain / Cas9 C-terminal domain / Cas9, topo homolgy domain / CRISPR-associated endonuclease Cas9 beta-hairpin domain / Topo homolgy domain in CRISPR-associated endonuclease Cas9 / Cas9 C-terminal domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / HNH endonuclease / HNH endonuclease ...CRISPR-associated endonuclease Cas9, beta-hairpin domain / Cas9 C-terminal domain / Cas9, topo homolgy domain / CRISPR-associated endonuclease Cas9 beta-hairpin domain / Topo homolgy domain in CRISPR-associated endonuclease Cas9 / Cas9 C-terminal domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / HNH endonuclease / HNH endonuclease / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / HNH nucleases / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease, Csn1 family
Similarity search - Component
Biological speciesAcidothermus cellulolyticus (bacteria)
Acidothermus cellulolyticus 11B (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.61 Å
AuthorsLi, H. / Das, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM099604 United States
CitationJournal: Nat Commun / Year: 2020
Title: The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9.
Authors: Das, A. / Hand, T.H. / Smith, C.L. / Wickline, E. / Zawrotny, M. / Li, H.
History
DepositionMar 28, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Jun 2, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 18, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease, Csn1 family
B: sgRNA (95-MER)
C: DNA (30-MER)
D: DNA (5'-D(*AP*TP*AP*CP*TP*TP*GP*GP*CP*G)-3')


Theoretical massNumber of molelcules
Total (without water)152,2554
Polymers152,2554
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16920 Å2
ΔGint-116 kcal/mol
Surface area60550 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.635, 111.143, 177.615
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein CRISPR-associated endonuclease, Csn1 family


Mass: 109589.148 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acidothermus cellulolyticus (strain ATCC 43068 / 11B) (bacteria)
Strain: ATCC 43068 / 11B / Gene: Acel_1951 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0LWB3
#2: RNA chain sgRNA (95-MER)


Mass: 30477.963 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Acidothermus cellulolyticus 11B (bacteria)
#3: DNA chain DNA (30-MER)


Mass: 9127.895 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(*AP*TP*AP*CP*TP*TP*GP*GP*CP*G)-3')


Mass: 3060.016 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.65 Å3/Da / Density % sol: 53.57 %
Crystal growTemperature: 303.15 K / Method: vapor diffusion, hanging drop
Details: 0.04 M Citric acid, 0.06 M Bis-tris propane and 15-20% polyethylene glycol 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9792 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Jul 27, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 3.61→177.62 Å / Num. obs: 252772 / % possible obs: 99.9 % / Redundancy: 13.1 % / CC1/2: 0.997 / Rmerge(I) obs: 0.198 / Rpim(I) all: 0.058 / Net I/σ(I): 8.3
Reflection shellResolution: 3.61→3.95 Å / Rmerge(I) obs: 3.629 / Mean I/σ(I) obs: 1 / Num. unique obs: 63055 / CC1/2: 0.554 / Rpim(I) all: 1.003

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Processing

Software
NameVersionClassification
PHENIX1.18rc4_3812refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
XDSdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6WBR

6wbr


Resolution: 3.61→94.22 Å / SU ML: 0.64 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 38.11 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3036 3570 10.05 %
Rwork0.2373 31953 -
obs0.2439 35523 99.04 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 376.41 Å2 / Biso mean: 192.494 Å2 / Biso min: 111.2 Å2
Refinement stepCycle: final / Resolution: 3.61→94.22 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7371 2760 0 0 10131
Num. residues----1069
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 26

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.61-3.660.39641270.391155128294
3.66-3.710.36551350.37221199133494
3.71-3.760.38551320.39321166129897
3.76-3.820.40661320.37221228136099
3.82-3.880.37441380.35741243138199
3.88-3.940.46521430.347612281371100
3.94-4.010.34081380.36421226136499
4.01-4.080.47121390.346312681407100
4.08-4.160.44011330.31481217135099
4.16-4.240.30821370.316712351372100
4.24-4.340.31331350.276712451380100
4.34-4.440.35181360.27441219135599
4.44-4.550.33271380.253712691407100
4.55-4.670.2721380.274312121350100
4.67-4.810.30161410.26051246138799
4.81-4.960.35111330.271612141347100
4.96-5.140.34611350.263712581393100
5.14-5.350.34311410.257612341375100
5.35-5.590.36711410.285312261367100
5.59-5.880.32051380.27412441382100
5.89-6.250.40671380.261512641402100
6.25-6.740.33691390.255812341373100
6.74-7.410.29751390.219612291368100
7.41-8.490.26171450.196412291374100
8.49-10.690.22241400.17161225136599
10.7-94.220.24131390.16051240137999

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