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- PDB-8wmm: Structure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate ... -

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Basic information

Entry
Database: PDB / ID: 8wmm
TitleStructure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate (20-nt complementary)
Components
  • NTS
  • PcrIIC1
  • TS
  • deadCbCas9
  • sgRNA
KeywordsDNA BINDING PROTEIN/DNA/RNA / Cas9 complex / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesChryseobacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 2.98 Å
AuthorsZhang, S. / Lin, S. / Liu, J.J.G.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32150018 China
National Natural Science Foundation of China (NSFC)32101195 China
CitationJournal: Nature / Year: 2024
Title: Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.
Authors: Shouyue Zhang / Ao Sun / Jing-Mei Qian / Shuo Lin / Wenjing Xing / Yun Yang / Han-Zhou Zhu / Xin-Yi Zhou / Yan-Shuo Guo / Yun Liu / Yu Meng / Shu-Lin Jin / Wenhao Song / Cheng-Ping Li / ...Authors: Shouyue Zhang / Ao Sun / Jing-Mei Qian / Shuo Lin / Wenjing Xing / Yun Yang / Han-Zhou Zhu / Xin-Yi Zhou / Yan-Shuo Guo / Yun Liu / Yu Meng / Shu-Lin Jin / Wenhao Song / Cheng-Ping Li / Zhaofu Li / Shuai Jin / Jian-Hua Wang / Meng-Qiu Dong / Caixia Gao / Chunlai Chen / Yang Bai / Jun-Jie Gogo Liu /
Abstract: The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA. As part of the ongoing struggle between phages and the bacterial ...The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel β-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.
History
DepositionOct 4, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: deadCbCas9
B: deadCbCas9
C: TS
D: sgRNA
F: TS
G: PcrIIC1
H: PcrIIC1
O: sgRNA
S: NTS
Y: NTS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)488,38912
Polymers488,34110
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 4 molecules ABGH

#1: Protein deadCbCas9


Mass: 170029.109 Da / Num. of mol.: 2 / Mutation: D9A; H837A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chryseobacterium (bacteria) / Production host: Escherichia coli (E. coli)
#4: Protein PcrIIC1


Mass: 15991.991 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chryseobacterium (bacteria) / Production host: Escherichia coli (E. coli)

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DNA chain , 2 types, 4 molecules CFSY

#2: DNA chain TS


Mass: 8450.413 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Chryseobacterium (bacteria)
#5: DNA chain NTS


Mass: 8762.670 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Chryseobacterium (bacteria)

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RNA chain / Non-polymers , 2 types, 4 molecules DO

#3: RNA chain sgRNA


Mass: 40936.227 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Chryseobacterium (bacteria)
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Structure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate (20-nt complementary)
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.4696 MDa / Experimental value: NO
Source (natural)Organism: Chryseobacterium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 150mM NaCl, 20mM Hepes (pH=7.5), 5mM MgCl2, 1mM Tcep, 0.1% Glycerol
Buffer componentConc.: 1 uM / Name: sodium chloride / Formula: NaCl
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 355919 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00229818
ELECTRON MICROSCOPYf_angle_d0.47141536
ELECTRON MICROSCOPYf_dihedral_angle_d13.5176286
ELECTRON MICROSCOPYf_chiral_restr0.0374720
ELECTRON MICROSCOPYf_plane_restr0.0034216

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