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- PDB-5meb: Crystal structure of yeast Cdt1 C-terminal domain -

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Basic information

Entry
Database: PDB / ID: 5meb
TitleCrystal structure of yeast Cdt1 C-terminal domain
ComponentsCell division cycle protein CDT1
KeywordsCELL CYCLE / Cdt1 / MCM / winged helix / yeast / DNA replication
Function / homology
Function and homology information


pre-replicative complex assembly involved in nuclear cell cycle DNA replication / nuclear pre-replicative complex / double-strand break repair via break-induced replication / regulation of DNA-templated DNA replication initiation / DNA replication origin binding / cell division / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
DNA replication factor Cdt1, C-terminal / DNA replication factor Cdt1, C-terminal WH domain superfamily / DNA replication factor Cdt1 C-terminal domain
Similarity search - Domain/homology
Cell division cycle protein CDT1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.8 Å
AuthorsPye, V.E. / Frigola, J. / Diffley, J.F.X. / Cherepanov, P.
CitationJournal: Nat Commun / Year: 2017
Title: Cdt1 stabilizes an open MCM ring for helicase loading.
Authors: Jordi Frigola / Jun He / Kerstin Kinkelin / Valerie E Pye / Ludovic Renault / Max E Douglas / Dirk Remus / Peter Cherepanov / Alessandro Costa / John F X Diffley /
Abstract: ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM ...ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2-5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2-5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC-Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.
History
DepositionNov 14, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 17, 2017Provider: repository / Type: Initial release
Revision 1.1Jul 5, 2017Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_volume ..._citation.country / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cell division cycle protein CDT1
B: Cell division cycle protein CDT1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,0624
Polymers25,8702
Non-polymers1922
Water5,747319
1
A: Cell division cycle protein CDT1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,1273
Polymers12,9351
Non-polymers1922
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Cell division cycle protein CDT1


Theoretical massNumber of molelcules
Total (without water)12,9351
Polymers12,9351
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)43.930, 85.870, 89.190
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Cell division cycle protein CDT1 / SIC1 indispensable protein 2 / Topoisomerase-A hypersensitive protein 11


Mass: 12935.186 Da / Num. of mol.: 2 / Fragment: UNP residues 495-604
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TAH11, CDT1, SID2, YJR046W, J1641 / Production host: Escherichia coli (E. coli) / References: UniProt: P47112
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 319 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.51 Å3/Da / Density % sol: 62.17 % / Description: 64.94
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 20% glycerol, 0.1M MES pH 6.5, 1.8M ammonium sulphate

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 0.97246 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Jun 8, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97246 Å / Relative weight: 1
ReflectionResolution: 1.8→45 Å / Num. obs: 31908 / % possible obs: 99.6 % / Redundancy: 18 % / Rmerge(I) obs: 0.11 / Net I/σ(I): 20.2
Reflection shellResolution: 1.8→1.85 Å / Redundancy: 18.4 % / Rmerge(I) obs: 1.91 / Mean I/σ(I) obs: 1.91 / % possible all: 98.9

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Processing

Software
NameVersionClassification
PHENIX1.8.4_1496refinement
XDSdata reduction
Aimlessdata scaling
PHENIXphenix.autosolvephasing
RefinementMethod to determine structure: SAD / Resolution: 1.8→44.595 Å / SU ML: 0.19 / Cross valid method: FREE R-VALUE / σ(F): 0.45 / Phase error: 21.1
RfactorNum. reflection% reflectionSelection details
Rfree0.21 3116 5.06 %random
Rwork0.1835 ---
obs0.1848 31905 99.62 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.8→44.595 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1762 0 10 319 2091
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0131877
X-RAY DIFFRACTIONf_angle_d1.2492543
X-RAY DIFFRACTIONf_dihedral_angle_d13.948734
X-RAY DIFFRACTIONf_chiral_restr0.061283
X-RAY DIFFRACTIONf_plane_restr0.005324
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.82810.29551460.28952633X-RAY DIFFRACTION99
1.8281-1.85810.29551370.28332537X-RAY DIFFRACTION100
1.8581-1.89020.28161380.26082604X-RAY DIFFRACTION100
1.8902-1.92450.30671420.25032558X-RAY DIFFRACTION99
1.9245-1.96150.24781580.22192604X-RAY DIFFRACTION100
1.9615-2.00160.21421410.22442581X-RAY DIFFRACTION99
2.0016-2.04510.20341410.20362600X-RAY DIFFRACTION100
2.0451-2.09270.21221310.18362594X-RAY DIFFRACTION99
2.0927-2.1450.22581380.18242578X-RAY DIFFRACTION100
2.145-2.2030.25381410.18752638X-RAY DIFFRACTION99
2.203-2.26780.21551350.18552588X-RAY DIFFRACTION100
2.2678-2.3410.22751350.18372598X-RAY DIFFRACTION100
2.341-2.42470.2771950.19312652X-RAY DIFFRACTION100
2.4247-2.52180.23111240.18762605X-RAY DIFFRACTION100
2.5218-2.63650.20571590.17722560X-RAY DIFFRACTION100
2.6365-2.77550.20241420.17532619X-RAY DIFFRACTION100
2.7755-2.94940.20751210.18412621X-RAY DIFFRACTION100
2.9494-3.1770.17721330.17782617X-RAY DIFFRACTION100
3.177-3.49660.20581420.16032574X-RAY DIFFRACTION100
3.4966-4.00230.1761680.15412612X-RAY DIFFRACTION100
4.0023-5.04140.17331300.15332608X-RAY DIFFRACTION100
5.0414-44.60870.2171500.20342579X-RAY DIFFRACTION99
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.06280.5218-0.04271.17360.44780.5511-0.0693-0.2601-0.21360.352-0.21550.2290.561-0.6042-0.00010.4178-0.03110.06660.4128-0.01810.298429.994354.681563.2003
25.02170.13110.6543.0478-0.55941.58610.0897-0.1081-0.19030.0456-0.0637-0.03270.0516-0.0414-0.00660.1355-0.0176-0.00760.14230.00180.133442.317159.910445.8245
30.992-0.1478-0.04781.3390.24260.3634-0.3124-0.2440.08440.69940.06490.2518-0.3976-0.2340.00060.3282-0.00130.01780.252-0.06130.321531.275347.754847.3732
40.6352-0.56830.20180.1829-0.12890.5880.0040.14750.1322-0.1458-0.12810.0153-0.1955-0.2123-0.00030.34880.03010.00070.32480.00720.267128.356330.065426.7729
54.3117-0.8668-0.51362.5186-0.49451.75430.09950.17810.0507-0.0441-0.0557-0.00090.00690.0092-0.00210.14440.01620.01050.1367-0.00220.120441.066125.461542.5307
60.7406-0.0303-0.18521.28370.57310.29830.05210.13790.2411-0.3483-0.29710.2720.0892-0.3733-0.00010.29450.0409-0.00890.2255-0.02810.350130.012737.144643.1725
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain A and resid 493:513)
2X-RAY DIFFRACTION2(chain A and resid 514:586)
3X-RAY DIFFRACTION3(chain A and resid 587:604)
4X-RAY DIFFRACTION4(chain B and resid 493:514)
5X-RAY DIFFRACTION5(chain B and resid 515:584)
6X-RAY DIFFRACTION6(chain B and resid 585:601)

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