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- PDB-1sxj: Crystal Structure of the Eukaryotic Clamp Loader (Replication Fac... -

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Entry
Database: PDB / ID: 1sxj
TitleCrystal Structure of the Eukaryotic Clamp Loader (Replication Factor C, RFC) Bound to the DNA Sliding Clamp (Proliferating Cell Nuclear Antigen, PCNA)
Components
  • (Activator 1 40 kDa ...) x 2
  • Activator 1 37 kDa subunit
  • Activator 1 41 kDa subunit
  • Activator 1 95 kDa subunit
  • Proliferating cell nuclear antigen
KeywordsREPLICATION / CLAMP LOADER / PROCESSIVITY CLAMP / DNA SLIDING CLAMP / AAA+ ATPASE / DNA POLYMERASE / DNA-BINDING PROTEIN
Function / homology
Function and homology information


DNA clamp unloading / Rad17 RFC-like complex / Elg1 RFC-like complex / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / DNA replication factor C complex / Ctf18 RFC-like complex / meiotic mismatch repair / DNA clamp loader activity / Processive synthesis on the lagging strand / Removal of the Flap Intermediate ...DNA clamp unloading / Rad17 RFC-like complex / Elg1 RFC-like complex / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / DNA replication factor C complex / Ctf18 RFC-like complex / meiotic mismatch repair / DNA clamp loader activity / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / Polymerase switching / positive regulation of DNA metabolic process / SUMOylation of DNA replication proteins / maintenance of DNA trinucleotide repeats / PCNA complex / establishment of mitotic sister chromatid cohesion / DNA replication checkpoint signaling / Activation of ATR in response to replication stress / lagging strand elongation / postreplication repair / sister chromatid cohesion / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / leading strand elongation / DNA polymerase processivity factor activity / error-free translesion synthesis / Gap-filling DNA repair synthesis and ligation in TC-NER / subtelomeric heterochromatin formation / mismatch repair / translesion synthesis / positive regulation of DNA replication / positive regulation of DNA repair / replication fork / DNA damage checkpoint signaling / nucleotide-excision repair / DNA-templated DNA replication / mitotic cell cycle / chromosome, telomeric region / cell division / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / identical protein binding / cytosol
Similarity search - Function
Zinc Finger, Delta Prime; domain 3 - #10 / Zinc Finger, Delta Prime; domain 3 / Replication factor C subunit 1 / DNA replication factor RFC1, C-terminal / Replication factor RFC1 C terminal domain / DNA Polymerase III; Chain A, domain 2 / DNA Polymerase III, subunit A, domain 2 / Replication factor C, C-terminal / Replication factor C C-terminal domain / : ...Zinc Finger, Delta Prime; domain 3 - #10 / Zinc Finger, Delta Prime; domain 3 / Replication factor C subunit 1 / DNA replication factor RFC1, C-terminal / Replication factor RFC1 C terminal domain / DNA Polymerase III; Chain A, domain 2 / DNA Polymerase III, subunit A, domain 2 / Replication factor C, C-terminal / Replication factor C C-terminal domain / : / DNA polymerase III, delta subunit / Box / Proliferating Cell Nuclear Antigen / Proliferating Cell Nuclear Antigen - #10 / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / Proliferating cell nuclear antigen signature 2. / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Proliferating cell nuclear antigen, PCNA / Proliferating cell nuclear antigen, PCNA, N-terminal / Proliferating cell nuclear antigen, PCNA, C-terminal / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-terminal domain / Helicase, Ruva Protein; domain 3 - #60 / : / BRCA1 C Terminus (BRCT) domain / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Helicase, Ruva Protein; domain 3 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Roll / Up-down Bundle / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Proliferating cell nuclear antigen / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 1 / Replication factor C subunit 4 / Replication factor C subunit 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.85 Å
AuthorsBowman, G.D. / O'Donnell, M. / Kuriyan, J.
CitationJournal: Nature / Year: 2004
Title: Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex.
Authors: Bowman, G.D. / O'Donnell, M. / Kuriyan, J.
History
DepositionMar 30, 2004Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 22, 2004Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Oct 27, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / struct_conn_type / struct_ref_seq_dif / struct_sheet / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn_type.id / _struct_ref_seq_dif.details / _struct_sheet.number_strands / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Aug 23, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.5Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2
Remark 999SEQUENCE The actual sequence of the region 697-747 (shown as UNK in SEQRES) is ...SEQUENCE The actual sequence of the region 697-747 (shown as UNK in SEQRES) is DKIGLRLDYLPTFRKRLLDPFLKQGADAISSVIEVMDDYYLTKEDWDSIME

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Activator 1 95 kDa subunit
B: Activator 1 37 kDa subunit
C: Activator 1 40 kDa subunit
D: Activator 1 41 kDa subunit
E: Activator 1 40 kDa subunit
F: Proliferating cell nuclear antigen
G: Proliferating cell nuclear antigen
H: Proliferating cell nuclear antigen
hetero molecules


Theoretical massNumber of molelcules
Total (without water)309,28217
Polymers306,6658
Non-polymers2,6179
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area37350 Å2
ΔGint-160 kcal/mol
Surface area104140 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.209, 110.481, 268.206
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 4 types, 6 molecules ABDFGH

#1: Protein Activator 1 95 kDa subunit / Replication factor C subunit 1 / Replication factor C1 / Cell division control protein 44


Mass: 56377.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC1, CDC44, YOR217W, YOR50-7 / Plasmid: pLANT / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P38630
#2: Protein Activator 1 37 kDa subunit / Replication factor C subunit 4 / Replication factor C4


Mass: 36171.977 Da / Num. of mol.: 1 / Mutation: R162Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC4, YOL094C, O0923 / Plasmid: pET11 / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P40339
#4: Protein Activator 1 41 kDa subunit / Replication factor C subunit 2 / Replication factor C2


Mass: 39765.410 Da / Num. of mol.: 1 / Mutation: R160Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC2, YJR068W, J1808 / Plasmid: pET11 / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P40348
#6: Protein Proliferating cell nuclear antigen / / PCNA


Mass: 32053.217 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL30, YBR088C, YBR0811 / Plasmid: pET28 / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P15873

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Activator 1 40 kDa ... , 2 types, 2 molecules CE

#3: Protein Activator 1 40 kDa subunit / Replication factor C subunit 3 / Replication factor C3


Mass: 38225.480 Da / Num. of mol.: 1 / Mutation: R165Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC3, YNL290W, N0533 / Plasmid: pET11 / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P38629
#5: Protein Activator 1 40 kDa subunit / Replication factor C subunit 5 / Replication factor C5


Mass: 39964.520 Da / Num. of mol.: 1 / Mutation: R184Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC5, YBR087W, YBR0810 / Plasmid: pLANT / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P38251

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Non-polymers , 3 types, 9 molecules

#7: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#8: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#9: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 2

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Sample preparation

CrystalDensity Matthews: 2.52 Å3/Da / Density % sol: 51.14 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 9
Details: PEG 3350, sodium chloride, CHES, pH 9.0, VAPOR DIFFUSION, HANGING DROP, temperature 292K
Crystal grow
*PLUS
Temperature: 20 ℃
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formula
130-50 mg/mlprotein1drop
2300 mM1reservoirNaCl
314.4-15.2 %(w/v)PEG33501reservoir
4100 mMCHES1reservoir
55 mMbeta-mercaptoethanol1reservoir
610 mMATP-gammaS1drop
710 mM1dropMgCl2
84 mMdithiothreitol1drop
92 mMTCEP1drop

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Data collection

Diffraction
IDMean temperature (K)Crystal-ID
11001
21001
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONALS 8.2.210.9464, 0.9798
SYNCHROTRONALS 8.2.221
Detector
TypeIDDetectorDate
ADSC QUANTUM 3151CCDJun 11, 2003
ADSC QUANTUM 3152CCDJul 9, 2003
Radiation
IDMonochromatorProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1Double crystal Si(111)MADMx-ray1
2Double crystal Si(111)SINGLE WAVELENGTHMx-ray1
Radiation wavelength
IDWavelength (Å)Relative weight
10.94641
20.97981
311
ReflectionResolution: 2.85→100 Å / Num. all: 139719 / Num. obs: 132895 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 9.1 % / Biso Wilson estimate: 41.4 Å2 / Rsym value: 0.09 / Net I/σ(I): 22.7
Reflection shellResolution: 2.85→2.95 Å / Redundancy: 5.4 % / Mean I/σ(I) obs: 2.7 / Num. unique all: 7230 / Rsym value: 0.481 / % possible all: 100
Reflection
*PLUS
Lowest resolution: 100 Å / % possible obs: 100 %

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Processing

Software
NameVersionClassification
CNS1.1refinement
HKL-2000data reduction
SCALEPACKdata scaling
SOLVEphasing
SHARPphasing
RefinementMethod to determine structure: MAD
Starting model: 1PLQ, 1JR3, 1NJF, 1IQP
Resolution: 2.85→48.81 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 122662.92 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
Details: IN CHAIN A, IT WAS NOT POSSIBLE TO UNAMBIGUOUSLY DETERMINE THE SEQUENCE REGISTER FOR RESIDUES 697 TO 747. THESE RESIDUES ARE THEREFORE DESIGNATED UNK TO INDICATE THIS AMBIGUITY. IN CHAIN E, ...Details: IN CHAIN A, IT WAS NOT POSSIBLE TO UNAMBIGUOUSLY DETERMINE THE SEQUENCE REGISTER FOR RESIDUES 697 TO 747. THESE RESIDUES ARE THEREFORE DESIGNATED UNK TO INDICATE THIS AMBIGUITY. IN CHAIN E, RESIDUES 86 TO 121 ARE GIVEN THE AMINO ACID TYPES THAT CORRESPOND TO THE GENE NUMBERING, HOWEVER THE SEQUENCE REGISTER MAY BE INCORRECT DUE TO A LACK OF DENSITY FOR FLANKING LOOPS.
RfactorNum. reflection% reflectionSelection details
Rfree0.306 6077 4.6 %RANDOM
Rwork0.251 ---
all0.251 132895 --
obs0.251 132895 95.1 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 37.1289 Å2 / ksol: 0.280894 e/Å3
Displacement parametersBiso mean: 91.7 Å2
Baniso -1Baniso -2Baniso -3
1--24.33 Å20 Å20 Å2
2--18.11 Å20 Å2
3---6.22 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.55 Å0.44 Å
Luzzati d res low-5 Å
Luzzati sigma a0.7 Å0.61 Å
Refinement stepCycle: LAST / Resolution: 2.85→48.81 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms19585 0 155 0 19740
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.005
X-RAY DIFFRACTIONc_angle_deg1.1
X-RAY DIFFRACTIONc_dihedral_angle_d21.8
X-RAY DIFFRACTIONc_improper_angle_d0.69
X-RAY DIFFRACTIONc_mcbond_it5.171.5
X-RAY DIFFRACTIONc_mcangle_it7.762
X-RAY DIFFRACTIONc_scbond_it8.672
X-RAY DIFFRACTIONc_scangle_it11.422.5
LS refinement shellResolution: 2.85→3.03 Å / Rfactor Rfree error: 0.014 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.429 896 4.5 %
Rwork0.398 18948 -
obs--85.3 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2ION.PARAMION.TOP
X-RAY DIFFRACTION3WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION4ATG_ADP_PAR.TXTATG_ADP_TOP.TXT
Software
*PLUS
Version: 1.1 / Classification: refinement
Refinement
*PLUS
σ(F): 0 / % reflection Rfree: 4.6 %
Solvent computation
*PLUS
Displacement parameters
*PLUS
Biso mean: 91.7 Å2
Refine LS restraints
*PLUS
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_angle_deg1.1
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg21.8
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.69
X-RAY DIFFRACTIONc_mcbond_it5.171.5
X-RAY DIFFRACTIONc_scbond_it8.672
X-RAY DIFFRACTIONc_mcangle_it7.762
X-RAY DIFFRACTIONc_scangle_it11.422.5
LS refinement shell
*PLUS
Rfactor Rfree: 0.429 / % reflection Rfree: 4.5 % / Rfactor Rwork: 0.398

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