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- PDB-5f9r: Crystal structure of catalytically-active Streptococcus pyogenes ... -

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Basic information

Entry
Database: PDB / ID: 5f9r
TitleCrystal structure of catalytically-active Streptococcus pyogenes CRISPR-Cas9 in complex with single-guided RNA and double-stranded DNA primed for target DNA cleavage
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (30-MER)
  • DNA (5'-D(P*AP*TP*GP*AP*GP*AP*CP*GP*CP*TP*GP*GP*AP*GP*TP*AP*CP*AP*C)-3')
  • RNA (116-MER)
KeywordsHYDROLASE/DNA/RNA / CRISPR / Cas9 / R-loop / genome engineering / HYDROLASE-DNA-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
Streptococcus pyogenes MGAS8232 (bacteria)
Lambdapapillomavirus 1
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 3.4 Å
AuthorsJiang, F. / Doudna, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Science / Year: 2016
Title: Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.
Authors: Fuguo Jiang / David W Taylor / Janice S Chen / Jack E Kornfeld / Kaihong Zhou / Aubri J Thompson / Eva Nogales / Jennifer A Doudna /
Abstract: Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding ...Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.
History
DepositionDec 10, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 27, 2016Provider: repository / Type: Initial release
Revision 1.1Feb 17, 2016Group: Database references
Revision 1.2Mar 9, 2016Group: Database references
Revision 1.3Sep 27, 2017Group: Author supporting evidence / Database references / Derived calculations
Category: citation / pdbx_audit_support / pdbx_struct_oper_list
Item: _citation.journal_id_CSD / _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.4Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: CRISPR-associated endonuclease Cas9/Csn1
A: RNA (116-MER)
C: DNA (30-MER)
D: DNA (5'-D(P*AP*TP*GP*AP*GP*AP*CP*GP*CP*TP*GP*GP*AP*GP*TP*AP*CP*AP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,7297
Polymers215,4404
Non-polymers2883
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area22860 Å2
ΔGint-172 kcal/mol
Surface area81390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)147.940, 230.100, 417.650
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number22
Space group name H-MF222

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Components

#1: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpyCas9


Mass: 158699.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#2: RNA chain RNA (116-MER)


Mass: 38292.645 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: RNA was prepared by in vitro transcription with T7 RNA polymerase
Source: (synth.) Streptococcus pyogenes MGAS8232 (bacteria)
#3: DNA chain DNA (30-MER)


Mass: 9085.841 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Lambdapapillomavirus 1
#4: DNA chain DNA (5'-D(P*AP*TP*GP*AP*GP*AP*CP*GP*CP*TP*GP*GP*AP*GP*TP*AP*CP*AP*C)-3')


Mass: 9362.040 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Lambdapapillomavirus 1
#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4

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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: 4.2 Å3/Da / Density % sol: 70.75 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 1.8 M ammonium sulfate, 100 mM Tris-HCl pH8.0 and 10 mM EDTA

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.116,0.98,0.957
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Aug 25, 2015
RadiationProtocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
11.1161
20.981
30.9571
ReflectionResolution: 3.4→119 Å / Num. obs: 48447 / % possible obs: 99 % / Redundancy: 3.8 % / Rsym value: 0.056 / Net I/σ(I): 6.7
Reflection shellResolution: 3.4→3.6 Å / Redundancy: 3.6 % / Rmerge(I) obs: 0.52 / Mean I/σ(I) obs: 1.9 / % possible all: 99.7

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
iMOSFLMdata reduction
SCALAdata scaling
PHENIXphasing
RefinementMethod to determine structure: MAD / Resolution: 3.4→69.725 Å / SU ML: 0.53 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 33.89 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2986 2401 4.96 %
Rwork0.2369 --
obs0.2399 48395 98.75 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.4→69.725 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10146 3491 15 0 13652
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0114243
X-RAY DIFFRACTIONf_angle_d1.48120153
X-RAY DIFFRACTIONf_dihedral_angle_d15.3355469
X-RAY DIFFRACTIONf_chiral_restr0.0682432
X-RAY DIFFRACTIONf_plane_restr0.0082010
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.4-3.46940.39581560.34562576X-RAY DIFFRACTION96
3.4694-3.54480.41761240.33472648X-RAY DIFFRACTION97
3.5448-3.62730.34941360.31162659X-RAY DIFFRACTION98
3.6273-3.7180.35291200.30272687X-RAY DIFFRACTION98
3.718-3.81850.38841540.29492676X-RAY DIFFRACTION99
3.8185-3.93090.31021290.29732678X-RAY DIFFRACTION99
3.9309-4.05770.371290.29132720X-RAY DIFFRACTION99
4.0577-4.20270.32621620.28892686X-RAY DIFFRACTION99
4.2027-4.3710.31831360.26022691X-RAY DIFFRACTION99
4.371-4.56990.32751570.25262710X-RAY DIFFRACTION100
4.5699-4.81080.29891410.23872704X-RAY DIFFRACTION99
4.8108-5.11210.30961460.23322734X-RAY DIFFRACTION100
5.1121-5.50670.27771380.23462736X-RAY DIFFRACTION100
5.5067-6.06050.33481400.23792735X-RAY DIFFRACTION100
6.0605-6.93680.28471380.2272773X-RAY DIFFRACTION100
6.9368-8.7370.23971500.19692747X-RAY DIFFRACTION99
8.737-69.73960.24061450.17532834X-RAY DIFFRACTION98

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