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- EMDB-37656: Structure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate ... -

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Entry
Database: EMDB / ID: EMD-37656
TitleStructure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate (20-nt complementary)
Map data
Sample
  • Complex: Structure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate (20-nt complementary)
    • Protein or peptide: deadCbCas9
    • DNA: TS
    • RNA: sgRNA
    • Protein or peptide: PcrIIC1
    • DNA: NTS
  • Ligand: MAGNESIUM ION
KeywordsCas9 complex / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex
Biological speciesChryseobacterium (bacteria)
Methodsingle particle reconstruction / Resolution: 2.98 Å
AuthorsZhang S / Lin S / Liu JJG
Funding support China, 2 items
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, 2023-
Header (metadata) releaseJun 5, 2024-
Map releaseJun 5, 2024-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37656.png

Downloads & links

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Map

FileDownload / File: emd_37656.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.0825 Å
Density
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.0017826913 - 2.0650342
Average (Standard dev.)0.0008398916 (±0.019536978)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 378.875 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_37656_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_37656_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 : Structure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate ...

EntireName: Structure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate (20-nt complementary)
Components
  • Complex: Structure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate (20-nt complementary)
    • Protein or peptide: deadCbCas9
    • DNA: TS
    • RNA: sgRNA
    • Protein or peptide: PcrIIC1
    • DNA: NTS
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Structure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate ...

SupramoleculeName: Structure of CbCas9-PcrIIC1 complex bound to 28-bp DNA substrate (20-nt complementary)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Chryseobacterium (bacteria)
Molecular weightTheoretical: 469.6 KDa

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Macromolecule #1: deadCbCas9

MacromoleculeName: deadCbCas9 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Chryseobacterium (bacteria)
Molecular weightTheoretical: 170.029109 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIKNILGLAL GTNSIGWALV KQDFENKQGE ILGMGSRIIP MSQDILGDFG KGNSVSQTAE RTKYRSVRRL RERFLLRRER LHRVLYILN FLPEHYASQI DFEKRLGKFK VETEPKLVWK NTDGQFSFLF QNSFNEMLED FKAAGQELKI PYDWTIYHLR K KAISQKIE ...String:
MIKNILGLAL GTNSIGWALV KQDFENKQGE ILGMGSRIIP MSQDILGDFG KGNSVSQTAE RTKYRSVRRL RERFLLRRER LHRVLYILN FLPEHYASQI DFEKRLGKFK VETEPKLVWK NTDGQFSFLF QNSFNEMLED FKAAGQELKI PYDWTIYHLR K KAISQKIE KEELAWILLN FNHKRGYYQL RGEDFEEEKD KTFVRLKVDR IVDSGENVKG KILYDVYFEN GWKYDKQVVK TE DWVDRTK EFIVSESILK NGETKRTFKA VDSEKDWIAI KTKTEQEIEH SHKTVGTYIY ETLLQNPKQK IKGKLVRTIE RKF YKEELR QILEKQKEFH QELQSDDLYN DCIRELYRNN EVHQLTLRKK DFVHLFMEDI IFYQRPLRSQ KSSVSNCTLE FRKY KGENG AEHTQYLKAI PKSNPYYQEF RLWQWIFNLN LYTKDNDENV TKVFLNTTQD FENLFEFLNT RKEVDQKALL KHFKL NEKT HRWNFVEDKK YPCNETKTMI SSRLDKVENI SDDFLTRDIE QKIWHIIYSV NDKVEYEKAL KSFARKHHLD ESSFFE AFR KFPPFKSEYG SFSEKAIKKL LPLMRLGKYW NYAEIDKYSR ERIQKIITGE YDENIKDKVR EKSVHLTIEN DFQGLQL WL AQYIVYGRHS EASMIGKWNS ANDLEVFLKD FKQHSLRNPI VEQVITETLR VVKDIWLKYG NGTKDFFNEI HIELGREM K LPADDRKKLT NQITENENTN LRIKALLAEM MNDHSVENVR PFSPMQQEIL KIYEDGVLKS DIEIEDDILK ISKTAQPSS SDLKRYKLWL EQKYKSPYTG QIIPLNKLFT PEYEIEAIIP QSRYFDDSFS NKIICESAVN KLKDNYIGLG FIKQFGGTII ELGFGKSVK VFDTEEYEDF VKKHYANNRG KRNKLLMEDI PEKMIERQLN DTRYISKYIS GILSNIVRVE DGSDEGVNSK N IVPGNGKI TTQLKQDWGL NDVWNDLILP RFERMNQLTN SKDFTAWNEN HQKFLPTVPI EFSKGFSKKR IDHRHHALDA LV IACATTD HVNLLNNQSA KSDTKRYDLK KKLMKFEKVV YHHTQTGEKI EREIPKQFLK PWEKFTVDAK HNLESIIVSF KQN LRVINK ATNYYEKYVE KDGTKNKERV EQAGTNWAIR KPMHKDTVSG KVDLPWVKVP KGKILTATRK SLDSSFDLKS IGSI TDTGI QKILKNYLAF KDGNPELAFS PEGIDDLNKN IEKYNDGKPH QPINKVRVFE LGSKFQVGQT GNKKGKYVEA AKGTN LFFA VYEDEKGKRS YETIPLNEVI ERQKQGLTSV PLENEKGSRL LFDLSPNDLV YVPEIDENID SNFVFSNLNK EKISRI YKV EKTSGTECYF VRQDIAYLIK QYDAKTKIGE LESQNKLQVT MTDDRIRITD TCVKINCDRL GNINFITKEK IKQIFNE FR

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Macromolecule #4: PcrIIC1

MacromoleculeName: PcrIIC1 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Chryseobacterium (bacteria)
Molecular weightTheoretical: 15.991991 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSLDKIAIDT NILLYAYDNR DLDKQDRAVE ILLKKPFVTQ LVVFEFIKVL ERRFKMDKKE ITKLTIKLLK EVIIPLSLHR DIYNYSQFL LQRYNFGLSD ILVLSDSILN NCTILLSEDM CNGMIVDKKL KIVNPFL

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Macromolecule #2: TS

MacromoleculeName: TS / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Chryseobacterium (bacteria)
Molecular weightTheoretical: 8.450413 KDa
SequenceString:
(DC)(DG)(DT)(DT)(DT)(DT)(DG)(DT)(DC)(DT) (DC)(DG)(DG)(DC)(DT)(DC)(DC)(DC)(DC)(DG) (DA)(DC)(DA)(DT)(DT)(DC)(DT)(DC)

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Macromolecule #5: NTS

MacromoleculeName: NTS / type: dna / ID: 5 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Chryseobacterium (bacteria)
Molecular weightTheoretical: 8.76267 KDa
SequenceString:
(DG)(DA)(DG)(DA)(DA)(DT)(DG)(DT)(DC)(DG) (DG)(DG)(DG)(DA)(DG)(DC)(DC)(DG)(DA)(DG) (DA)(DC)(DA)(DA)(DA)(DA)(DC)(DG)

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Macromolecule #3: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 3 / Number of copies: 2
Source (natural)Organism: Chryseobacterium (bacteria)
Molecular weightTheoretical: 40.936227 KDa
SequenceString:
GAGAAUGUCG GGGAGCCGAG GUUGUGAAUU GCUUUCAAAA AUUAUUGAGA AAUAAUUUUG AAAAGCAAUU CACAAUAAGG AUUAUUCCG UUGUGAAAAC AUUCAAGGCG GGGCAACUCG CCUUUUUU

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Component - Concentration: 1.0 uM / Component - Formula: NaCl / Component - Name: sodium chloride
Details: 150mM NaCl, 20mM Hepes (pH=7.5), 5mM MgCl2, 1mM Tcep, 0.1% Glycerol

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 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: 1.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 81000
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

Startup modelType of model: NONE / Details: Ab-initial in cryosparc
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 355919
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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