[English] 日本語
Yorodumi- EMDB-5625: Architecture of a helicase loading intermediate containing ORC-Cd... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5625 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Architecture of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 on DNA reveals similarity to DNA polymerase clamp loading complexes | |||||||||
Map data | Reconstruction of ORC-Cdc6-Cdt1-Mcm2-7 complex at DNA origin in ATPrS | |||||||||
Sample |
| |||||||||
Keywords | PreRC / ORC / Cdc6 / Cdt1 / Mcm2-7 | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 14.0 Å | |||||||||
Authors | Sun J / Evrin C / Samel S / Fernandez-Cid A / Riera A / Kawakami H / Stillman B / Speck C / Li H | |||||||||
Citation | Journal: Nat Struct Mol Biol / Year: 2013 Title: Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA. Authors: Jingchuan Sun / Cecile Evrin / Stefan A Samel / Alejandra Fernández-Cid / Alberto Riera / Hironori Kawakami / Bruce Stillman / Christian Speck / Huilin Li / Abstract: In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using ...In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action. | |||||||||
History |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5625.map.gz | 634.4 KB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-5625-v30.xml emd-5625.xml | 10.1 KB 10.1 KB | Display Display | EMDB header |
Images | emd_5625_1.png | 112.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5625 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5625 | HTTPS FTP |
-Validation report
Summary document | emd_5625_validation.pdf.gz | 78.7 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_5625_full_validation.pdf.gz | 77.8 KB | Display | |
Data in XML | emd_5625_validation.xml.gz | 493 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5625 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5625 | HTTPS FTP |
-Related structure data
Similar structure data |
---|
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|
-Map
File | Download / File: emd_5625.map.gz / Format: CCP4 / Size: 670.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Reconstruction of ORC-Cdc6-Cdt1-Mcm2-7 complex at DNA origin in ATPrS | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.23 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
-Entire : ORC-Cdc6-Cdt1-Mcm2-7 complex
Entire | Name: ORC-Cdc6-Cdt1-Mcm2-7 complex |
---|---|
Components |
|
-Supramolecule #1000: ORC-Cdc6-Cdt1-Mcm2-7 complex
Supramolecule | Name: ORC-Cdc6-Cdt1-Mcm2-7 complex / type: sample / ID: 1000 / Number unique components: 14 |
---|---|
Molecular weight | Theoretical: 1.1 MDa |
-Macromolecule #1: ORC-Cdc6-Cdt1-Mcm2-7 complex
Macromolecule | Name: ORC-Cdc6-Cdt1-Mcm2-7 complex / type: protein_or_peptide / ID: 1 / Name.synonym: OCCM / Number of copies: 1 / Recombinant expression: Yes / Database: NCBI |
---|---|
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast |
Molecular weight | Theoretical: 1.1 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
---|---|
Buffer | pH: 7.5 / Details: 50 mM HEPES-KOH |
Grid | Details: thin carbon layer on holey carbon, glow discharged |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK I Method: A thin carbon layer was floated onto holey carbon and glow discharged. 2.8 uL sample was added and after 30 seconds was blotted for 5 seconds and plunge-frozen. |
-Electron microscopy
Microscope | JEOL 2010F |
---|---|
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 400K magnification. |
Date | Mar 8, 2010 |
Image recording | Category: FILM / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Number real images: 800 / Average electron dose: 15 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.4 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 60000 |
Sample stage | Specimen holder: liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
Details | Particles were selected with e2boxer.py in swarm mode. Additional good particles were selected and bad particles were removed manually. After structural factor and CTF correction in EMAN1, the phase-flipped particles were normalized (edgenorm, hp=1) and pooled. Refine2d.py was run and particles in bad class averages were removed. Initial models were made using EMAN2, but 3D refinement was performed using EMAN1.8 without ctfc or ctfcw. |
---|---|
CTF correction | Details: each particle set by ctfit |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1, EMAN2 / Number images used: 92049 |
Final two d classification | Number classes: 1104 |