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- EMDB-38058: Structure of DNA-bound GajA dimer (focused refinement) -

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Basic information

Entry
Database: EMDB / ID: EMD-38058
TitleStructure of DNA-bound GajA dimer (focused refinement)
Map data
Sample
  • Complex: Binary complex of GajA with dsDNA
    • Protein or peptide: Endonuclease GajA
    • DNA: DNA (5'-D(*TP*TP*TP*AP*AP*TP*AP*AP*CP*CP*CP*GP*GP*TP*TP*AP*TP*TP*TP*TP*T)-3')
    • DNA: DNA (5'-D(*AP*AP*AP*AP*AP*TP*AP*AP*CP*CP*GP*GP*GP*TP*TP*AP*TP*TP*AP*AP*A)-3')
  • Ligand: CALCIUM ION
Keywordsendonuclease / Complex / DNA BINDING / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / metal ion binding
Similarity search - Function
AAA domain, group 15 / : / Overcoming lysogenization defect protein-like, TOPRIM domain / OLD protein-like, TOPRIM domain / AAA ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesBacillus cereus VD045 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsLi J / Wang Z / Wang L
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nature / Year: 2024
Title: Structures and activation mechanism of the Gabija anti-phage system.
Authors: Jing Li / Rui Cheng / Zhiming Wang / Wuliu Yuan / Jun Xiao / Xinyuan Zhao / Xinran Du / Shiyu Xia / Lianrong Wang / Bin Zhu / Longfei Wang /
Abstract: Prokaryotes have evolved intricate innate immune systems against phage infection. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB. GajA ...Prokaryotes have evolved intricate innate immune systems against phage infection. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB. GajA functions as a DNA endonuclease that is inactive in the presence of ATP. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.
History
DepositionNov 16, 2023-
Header (metadata) releaseFeb 28, 2024-
Map releaseFeb 28, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38058.png

Downloads & links

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Map

FileDownload / File: emd_38058.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 384 pix.
= 322.56 Å		0.84 Å/pix.
x 384 pix.
= 322.56 Å		0.84 Å/pix.
x 384 pix.
= 322.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-1.2548469 - 2.058265
Average (Standard dev.)0.00020495552 (±0.030691622)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 322.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_38058_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_38058_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 : Binary complex of GajA with dsDNA

EntireName: Binary complex of GajA with dsDNA
Components
  • Complex: Binary complex of GajA with dsDNA
    • Protein or peptide: Endonuclease GajA
    • DNA: DNA (5'-D(*TP*TP*TP*AP*AP*TP*AP*AP*CP*CP*CP*GP*GP*TP*TP*AP*TP*TP*TP*TP*T)-3')
    • DNA: DNA (5'-D(*AP*AP*AP*AP*AP*TP*AP*AP*CP*CP*GP*GP*GP*TP*TP*AP*TP*TP*AP*AP*A)-3')
  • Ligand: CALCIUM ION

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Supramolecule #1: Binary complex of GajA with dsDNA

SupramoleculeName: Binary complex of GajA with dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Bacillus cereus VD045 (bacteria)

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Macromolecule #1: Endonuclease GajA

MacromoleculeName: Endonuclease GajA / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Bacillus cereus VD045 (bacteria)
Molecular weightTheoretical: 67.079469 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKFSNITIKN FRNFEKVNIN LDNKNVIFGM NDIGKTNFLY ALRFLLDKEI RKFGFNKSDY HKHDTSKKIE IILTLDLSNY EKDEDTKKL ISVVKGARTS ANADVFYIAL ESKYDDKELY GNIILKWGSE LDNLIDIPGR GNINALDNVF KVIYINPLVD L DKLFAQNK ...String:
MKFSNITIKN FRNFEKVNIN LDNKNVIFGM NDIGKTNFLY ALRFLLDKEI RKFGFNKSDY HKHDTSKKIE IILTLDLSNY EKDEDTKKL ISVVKGARTS ANADVFYIAL ESKYDDKELY GNIILKWGSE LDNLIDIPGR GNINALDNVF KVIYINPLVD L DKLFAQNK KYIFEESQGN ESDEGILNNI KSLTDQVNQQ IGEMTIIKGF QQEITSEYRS LKKEEVSIEL KSEMAIKGFF SD IIPYIKK DGDSNYYPTS GDGRRKMLSY SIYNYLAKKK YEDKIVIYLI EEPEISLHRS MQIALSKQLF EQSTYKYFFL STH SPELLY EMDNTRLIRV HSTEKVVCSS HMYNVEEAYG SVKKKLNKAL SSALFAERVL LIEGPSEKIL FEKVLDEVEP EYEL NGGFL LEVGGTYFNH YVCTLNDLGI THIIKTDNDL KSKKGKKGVY ELLGLNRCLN LLGRENLDEI TIDIPEDIKG KKKKE RLNE RKKEIFKQYK NEVGEFLGER IYLSEIDLEN DLYSAIGESM KRIFENEDPV HYLQKSKLFN MVELVNNLST KDCFDV FEH EKFACLKELV GSDRG

UniProtKB: Endonuclease GajA

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Macromolecule #2: DNA (5'-D(*TP*TP*TP*AP*AP*TP*AP*AP*CP*CP*CP*GP*GP*TP*TP*AP*TP*TP*...

MacromoleculeName: DNA (5'-D(*TP*TP*TP*AP*AP*TP*AP*AP*CP*CP*CP*GP*GP*TP*TP*AP*TP*TP*TP*TP*T)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.393154 KDa
SequenceString:
(DT)(DT)(DT)(DA)(DA)(DT)(DA)(DA)(DC)(DC) (DC)(DG)(DG)(DT)(DT)(DA)(DT)(DT)(DT)(DT) (DT)

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Macromolecule #3: DNA (5'-D(*AP*AP*AP*AP*AP*TP*AP*AP*CP*CP*GP*GP*GP*TP*TP*AP*TP*TP*...

MacromoleculeName: DNA (5'-D(*AP*AP*AP*AP*AP*TP*AP*AP*CP*CP*GP*GP*GP*TP*TP*AP*TP*TP*AP*AP*A)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.487263 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DT)(DA)(DA)(DC)(DC) (DG)(DG)(DG)(DT)(DT)(DA)(DT)(DT)(DA)(DA) (DA)

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.4000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 570317
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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