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- EMDB-20455: Cryo-EM structure of HzTransib/nicked TIR substrate DNA hairpin f... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-20455 | |||||||||
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Title | Cryo-EM structure of HzTransib/nicked TIR substrate DNA hairpin forming complex (HFC) | |||||||||
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![]() | RAG-like transposase / DDE family enzyme / Transib / Terminal inverted repeat. / RECOMBINATION / recombination-dna complex | |||||||||
Function / homology | metal ion binding / Putative DNA-mediated transposase![]() | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.6 Å | |||||||||
![]() | Liu C / Schatz DG / Yang Y | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Structures of a RAG-like transposase during cut-and-paste transposition. Authors: Chang Liu / Yang Yang / David G Schatz / ![]() Abstract: Transposons have had a pivotal role in genome evolution and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase, an essential component of the adaptive immune system in jawed ...Transposons have had a pivotal role in genome evolution and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase, an essential component of the adaptive immune system in jawed vertebrates. Here we report one crystal structure and five cryo-electron microscopy structures of Transib, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0-4.6 Å. These structures reveal a butterfly-shaped complex that undergoes two cycles of marked conformational changes in which the 'wings' of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA and close again to capture and attack target DNA. Transib possesses unique structural elements that compensate for the absence of a RAG2 partner, including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help to control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the detailed reaction pathway of a eukaryotic cut-and-paste transposase and illuminate some of the earliest steps in the evolution of the RAG recombinase. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 28.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 18.3 KB 18.3 KB | Display Display | ![]() |
Images | ![]() | 198.8 KB | ||
Filedesc metadata | ![]() | 6.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6pqxMC ![]() 6pqnC ![]() 6pqrC ![]() 6pquC ![]() 6pqyC ![]() 6pr5C C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.05 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Hairpin forming complex of HzTransib with nicked TIR substrate DNA
Entire | Name: Hairpin forming complex of HzTransib with nicked TIR substrate DNA |
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Components |
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-Supramolecule #1: Hairpin forming complex of HzTransib with nicked TIR substrate DNA
Supramolecule | Name: Hairpin forming complex of HzTransib with nicked TIR substrate DNA type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: DNA-mediated transposase
Macromolecule | Name: DNA-mediated transposase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 56.582734 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: KPAPSTIFSP EKALGLLLSL KLSKWQYITL RETTIREGSK EIYPSYYKVQ KAKLQCYPPK AFVAVTDSSA KIALQALLDL TVNRIFETI RSPDAIQNKQ LILISKWGFD GASNQSRYKQ NIESGQGDSS IFMTSLVPLK LTADGDTVWV NPKPCSPMYC R PVQFSFVK ...String: KPAPSTIFSP EKALGLLLSL KLSKWQYITL RETTIREGSK EIYPSYYKVQ KAKLQCYPPK AFVAVTDSSA KIALQALLDL TVNRIFETI RSPDAIQNKQ LILISKWGFD GASNQSRYKQ NIESGQGDSS IFMTSLVPLK LTADGDTVWV NPKPCSPMYC R PVQFSFVK ETKDVVINEK TAMDDEIEAL VPSKCQGHEI SHKLMMTMID GKICTYLSEA KSNAACYLCL AKPTEMSKLD VI ASKTISS GVYEFGLSTL HARINVMECL LHIAYRLDFK KWSARGEGHQ ELLHSRKKLI QDRFKDDLNL LIDIVKQGSG TTN DGNTAR RFFEFPDKTA AITGLDEDLI RRFSVILQAI TSGEIIDVPK FKEYARTTAE KYVELYDWYY MSSTVHKLLI HGGD IIAEN AIVPIGSLSE EASEARNKDF RRFREHHSRK KSRQASNEDI LNMLIISSDP LISFTRPKLD AHKRQTYFKE TVELL QLQD QEAPTEFHHH HHH UniProtKB: Putative DNA-mediated transposase |
-Macromolecule #2: DNA (5'-D(P*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*T)-3')
Macromolecule | Name: DNA (5'-D(P*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*T)-3') type: dna / ID: 2 / Number of copies: 2 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 4.889177 KDa |
Sequence | String: (DG)(DA)(DT)(DC)(DT)(DG)(DG)(DC)(DC)(DT) (DA)(DG)(DA)(DT)(DC)(DT) |
-Macromolecule #3: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
Macromolecule | Name: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3') type: dna / ID: 3 / Number of copies: 2 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 4.956244 KDa |
Sequence | String: (DC)(DA)(DC)(DG)(DG)(DT)(DG)(DG)(DA)(DT) (DC)(DG)(DA)(DA)(DA)(DA) |
-Macromolecule #4: TIR substrate DNA transferred strand
Macromolecule | Name: TIR substrate DNA transferred strand / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 9.792304 KDa |
Sequence | String: (DT)(DT)(DT)(DT)(DC)(DG)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DG)(DT)(DG)(DA)(DG)(DA)(DT) (DC)(DT)(DA)(DG)(DG)(DC)(DC)(DA)(DG) (DA)(DT)(DC) |
-Macromolecule #5: CALCIUM ION
Macromolecule | Name: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: CA |
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Molecular weight | Theoretical: 40.078 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.3 mg/mL | |||||||||||||||
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Buffer | pH: 7.5 Component:
Details: Solutions were made fresh from concentrated and filtered to avoid microbial contamination. | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 296 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 3 seconds before plunging. | |||||||||||||||
Details | Recombinantly expressed HzTransib transposase was mixed with chemically synthesized TIR substrate DNA. The complex was further purified on size-exclusion chromatography column. The final complex was monodisperse. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Details | Preliminary grid screening was performed manually. |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Average exposure time: 8.0 sec. / Average electron dose: 52.2 e/Å2 Details: Images were collected in movie-mode at 5 frames per second. |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 130000 |
Sample stage | Specimen 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
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A / Chain - Residue range: 21-501 / Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Details | Initial local fitting was done using UCSF Chimera, then manually adjusted and rebuilt in Coot. Final model was refined using Phenix real-space refinement. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient |
Output model | ![]() PDB-6pqx: |