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- PDB-5mec: Crystal structure of yeast Cdt1 middle domain (residues 294-433) -

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Basic information

Entry
Database: PDB / ID: 5mec
TitleCrystal structure of yeast Cdt1 middle domain (residues 294-433)
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 / MOLECULAR REPLACEMENT / Resolution: 2.13 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
Revision 1.2Jan 17, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cell division cycle protein CDT1


Theoretical massNumber of molelcules
Total (without water)19,2801
Polymers19,2801
Non-polymers00
Water1,09961
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area8520 Å2
MethodPISA
Unit cell
Length a, b, c (Å)45.130, 46.330, 75.360
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: 19280.357 Da / Num. of mol.: 1 / Fragment: UNP residues 272-438
Source method: isolated from a genetically manipulated source
Details: Residues 294-433 seen in the crystal structure, with exception of one loop which is not visible. Sequence provided is that which was expressed.
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TAH11, CDT1, SID2, YJR046W, J1641 / Production host: Escherichia coli (E. coli) / References: UniProt: P47112
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 61 / 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: 2.61 Å3/Da / Density % sol: 52.85 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.5 / Details: 0.1M BTP pH 5.5, 2M ammonium sulphate / PH range: 5.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Apr 20, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.13→39.47 Å / Num. obs: 9296 / % possible obs: 99.5 % / Redundancy: 6.9 % / Rmerge(I) obs: 0.078 / Net I/σ(I): 15
Reflection shellResolution: 2.13→2.19 Å / Redundancy: 7.3 % / Rmerge(I) obs: 0.62 / Mean I/σ(I) obs: 3.3 / % possible all: 99.2

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Processing

Software
NameVersionClassification
PHENIX1.8.4_1496refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2WVR chain C, residues 188-315 truncated to polyALA

2wvr


Resolution: 2.13→39.468 Å / SU ML: 0.3 / Cross valid method: FREE R-VALUE / σ(F): 1.12 / Phase error: 30.79
RfactorNum. reflection% reflectionSelection details
Rfree0.244 808 4.76 %Random selection
Rwork0.1965 ---
obs0.1989 9255 99.3 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 51 Å2
Refinement stepCycle: LAST / Resolution: 2.13→39.468 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1057 0 0 61 1118
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0021110
X-RAY DIFFRACTIONf_angle_d0.5721507
X-RAY DIFFRACTIONf_dihedral_angle_d10.977440
X-RAY DIFFRACTIONf_chiral_restr0.024178
X-RAY DIFFRACTIONf_plane_restr0.003189
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1301-2.26360.36731480.26982679X-RAY DIFFRACTION99
2.2636-2.43830.28011260.25282687X-RAY DIFFRACTION99
2.4383-2.68360.29691340.22732698X-RAY DIFFRACTION99
2.6836-3.07180.34051120.23462715X-RAY DIFFRACTION99
3.0718-3.86970.22141350.18652708X-RAY DIFFRACTION100
3.8697-39.47470.19561530.15922679X-RAY DIFFRACTION100
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)
10.65750.1672-0.20030.4433-0.27990.1665-0.063-0.0009-0.0564-0.05690.01710.06040.0891-0.18-0.00010.2866-0.0158-0.00210.215-0.01290.290312.59584.9910.1452
20.41930.19350.10760.2843-0.21320.32870.0377-0.36550.29890.0963-0.1448-0.1076-0.2854-0.2631-00.31830.02750.02050.36-0.03420.293315.11398.145319.3607
30.4070.1301-0.36630.1141-0.18650.35110.2468-0.35550.01450.1654-0.1041-0.03690.2354-0.73510.00070.4098-0.00150.01050.5220.00720.33695.75164.762816.674
40.75130.7826-0.53351.71270.05090.7977-0.2714-0.15180.3965-0.10550.1190.7175-0.1774-0.57820.02330.4170.19650.05130.4637-0.05460.5114.490116.068812.2084
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain A and resid 294:338)
2X-RAY DIFFRACTION2(chain A and resid 339:375)
3X-RAY DIFFRACTION3(chain A and resid 376:417)
4X-RAY DIFFRACTION4(chain A and resid 418:433)

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