+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-22453 | |||||||||
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Title | Adeno-Associated Virus 2 Rep68 HD Hexamer-ssDNA with ATPgS | |||||||||
Map data | Local refinement map of helicase domain | |||||||||
Sample |
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Keywords | AAV / Protein-DNA / AAA+ / SF3 / HUH / VIRAL PROTEIN / VIRAL PROTEIN-DNA complex | |||||||||
Function / homology | Function and homology information symbiont-mediated arrest of host cell cycle during G2/M transition / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / viral DNA genome replication / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / endonuclease activity / DNA replication / DNA helicase / host cell nucleus / ATP hydrolysis activity ...symbiont-mediated arrest of host cell cycle during G2/M transition / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / viral DNA genome replication / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / endonuclease activity / DNA replication / DNA helicase / host cell nucleus / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding Similarity search - Function | |||||||||
Biological species | Adeno-associated virus - 2 / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 5.2 Å | |||||||||
Authors | Escalante CR | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Nucleic Acids Res / Year: 2020 Title: The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states. Authors: Vishaka Santosh / Faik N Musayev / Rahul Jaiswal / Francisco Zárate-Pérez / Bram Vandewinkel / Caroline Dierckx / Molly Endicott / Kamyar Sharifi / Kelly Dryden / Els Henckaerts / Carlos R Escalante / Abstract: The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and ...The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_22453.map.gz | 96.3 MB | EMDB map data format | |
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Header (meta data) | emd-22453-v30.xml emd-22453.xml | 13.5 KB 13.5 KB | Display Display | EMDB header |
Images | emd_22453.png | 122.1 KB | ||
Filedesc metadata | emd-22453.cif.gz | 6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-22453 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-22453 | HTTPS FTP |
-Validation report
Summary document | emd_22453_validation.pdf.gz | 492 KB | Display | EMDB validaton report |
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Full document | emd_22453_full_validation.pdf.gz | 491.5 KB | Display | |
Data in XML | emd_22453_validation.xml.gz | 6.6 KB | Display | |
Data in CIF | emd_22453_validation.cif.gz | 7.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-22453 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-22453 | HTTPS FTP |
-Related structure data
Related structure data | 7jsgMC 6xb8C 7jseC 7jsfC 7jshC 7jsiC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_22453.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Local refinement map of helicase domain | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.073 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Rep68 Helicase in complex with ssDNA (poly-dT)
Entire | Name: Rep68 Helicase in complex with ssDNA (poly-dT) |
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Components |
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-Supramolecule #1: Rep68 Helicase in complex with ssDNA (poly-dT)
Supramolecule | Name: Rep68 Helicase in complex with ssDNA (poly-dT) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Heptameric complex model of helicase domain |
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Source (natural) | Organism: Adeno-associated virus - 2 |
Molecular weight | Theoretical: 424.5 KDa |
-Macromolecule #1: Protein Rep68
Macromolecule | Name: Protein Rep68 / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO / EC number: DNA helicase |
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Source (natural) | Organism: Adeno-associated virus - 2 |
Molecular weight | Theoretical: 60.908703 KDa |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: GPPGFYEIVI KVPSDLDEHL PGISDSFVNW VAEKEWELPP DSDMDLNLIE QAPLTVAEKL QRDFLTEWRR VSKAPEALFF VQFEKGESY FHMHVLVETT GVKSMVLGRF LSQIREKLIQ RIYRGIEPTL PNWFAVTKTR NGAGGGNKVV DESYIPNYLL P KTQPELQW ...String: GPPGFYEIVI KVPSDLDEHL PGISDSFVNW VAEKEWELPP DSDMDLNLIE QAPLTVAEKL QRDFLTEWRR VSKAPEALFF VQFEKGESY FHMHVLVETT GVKSMVLGRF LSQIREKLIQ RIYRGIEPTL PNWFAVTKTR NGAGGGNKVV DESYIPNYLL P KTQPELQW AWTNMEQYLS ACLNLTERKR LVAQHLTHVS QTQEQNKENQ NPNSDAPVIR SKTSARYMEL VGWLVDKGIT SE KQWIQED QASYISFNAA SNSRSQIKAA LDNAGKIMSL TKTAPDYLVG QQPVEDISSN RIYKILELNG YDPQYAASVF LGW ATKKFG KRNTIWLFGP ATTGKTNIAE AIAHTVPFYG CVNWTNENFP FNDCVDKMVI WWEEGKMTAK VVESAKAILG GSKV RVDQK CKSSAQIDPT PVIVTSNTNM CAVIDGNSTT FEHQQPLQDR MFKFELTRRL DHDFGKVTKQ EVKDFFRWAK DHVVE VEHE FYVKKGGAKK RPAPSDADIS EPKRVRESVA QPSTSDAEAS INYADRLARG HSL UniProtKB: Protein Rep68 |
-Macromolecule #2: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
Macromolecule | Name: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3') type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 5.12632 KDa |
Sequence | String: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.75 mg/mL | ||||||||||||||||||
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Buffer | pH: 7.9 Component:
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Grid | Model: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AMYLAMINE | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 4555 / Average electron dose: 55.46 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Protocol: RIGID BODY FIT |
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Output model | PDB-7jsg: |