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- PDB-4bpx: Crystal structure of human primase in complex with the primase- b... -

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Basic information

Entry
Database: PDB / ID: 4bpx
TitleCrystal structure of human primase in complex with the primase- binding motif of DNA polymerase alpha
Components
  • DNA POLYMERASE ALPHA CATALYTIC SUBUNIT, DNA PRIMASE LARGE SUBUNIT
  • DNA PRIMASE SMALL SUBUNIT
KeywordsTRANSFERASE / DNA REPLICATION / FUSION PROTEIN / CHIMERA
Function / homology
Function and homology information


: / DNA primase AEP / ribonucleotide binding / DNA replication initiation / Telomere C-strand synthesis initiation / DNA/RNA hybrid binding / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production / alpha DNA polymerase:primase complex / Polymerase switching ...: / DNA primase AEP / ribonucleotide binding / DNA replication initiation / Telomere C-strand synthesis initiation / DNA/RNA hybrid binding / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production / alpha DNA polymerase:primase complex / Polymerase switching / : / Processive synthesis on the lagging strand / DNA replication, synthesis of primer / lagging strand elongation / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere / mitotic DNA replication initiation / DNA synthesis involved in DNA repair / DNA strand elongation involved in DNA replication / leading strand elongation / G1/S-Specific Transcription / DNA replication origin binding / Activation of the pre-replicative complex / DNA replication initiation / Defective pyroptosis / double-strand break repair via nonhomologous end joining / nuclear matrix / nuclear envelope / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / DNA repair / nucleotide binding / chromatin binding / protein kinase binding / chromatin / nucleolus / magnesium ion binding / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / membrane / cytosol
Similarity search - Function
Transcription Elongation Factor S-II; Chain A - #80 / DNA primase, PRIM domain / DNA primase, PRIM domain / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily ...Transcription Elongation Factor S-II; Chain A - #80 / DNA primase, PRIM domain / DNA primase, PRIM domain / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / Eukaryotic and archaeal DNA primase, large subunit / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA polymerase alpha catalytic subunit, catalytic domain / Transcription Elongation Factor S-II; Chain A / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily / Alpha-Beta Complex / Up-down Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA polymerase alpha catalytic subunit / DNA primase small subunit / DNA primase large subunit
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.4 Å
AuthorsKilkenny, M.L. / Perera, R.L. / Pellegrini, L.
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2013
Title: Structures of Human Primase Reveal Design of Nucleotide Elongation Site and Mode of Pol Alpha Tethering
Authors: Kilkenny, M.L. / Longo, M. / Perera, R.L. / Pellegrini, L.
History
DepositionMay 28, 2013Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 25, 2013Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2013Group: Database references
Revision 1.2Sep 13, 2017Group: Data collection / Category: diffrn_detector / Item: _diffrn_detector.type
Revision 2.0Dec 20, 2023Group: Atomic model / Data collection ...Atomic model / Data collection / Database references / Derived calculations / Other / Refinement description
Category: atom_site / chem_comp_atom ...atom_site / chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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_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_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA PRIMASE SMALL SUBUNIT
B: DNA POLYMERASE ALPHA CATALYTIC SUBUNIT, DNA PRIMASE LARGE SUBUNIT
C: DNA PRIMASE SMALL SUBUNIT
D: DNA POLYMERASE ALPHA CATALYTIC SUBUNIT, DNA PRIMASE LARGE SUBUNIT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,8956
Polymers161,7644
Non-polymers1312
Water00
1
C: DNA PRIMASE SMALL SUBUNIT
D: DNA POLYMERASE ALPHA CATALYTIC SUBUNIT, DNA PRIMASE LARGE SUBUNIT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)80,9473
Polymers80,8822
Non-polymers651
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1590 Å2
ΔGint-7.9 kcal/mol
Surface area30360 Å2
MethodPISA
2
A: DNA PRIMASE SMALL SUBUNIT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,1732
Polymers50,1081
Non-polymers651
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
3
B: DNA POLYMERASE ALPHA CATALYTIC SUBUNIT, DNA PRIMASE LARGE SUBUNIT


Theoretical massNumber of molelcules
Total (without water)30,7741
Polymers30,7741
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)120.680, 70.701, 127.160
Angle α, β, γ (deg.)90.00, 105.83, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
22
/ NCS ensembles :
ID
1
2

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Components

#1: Protein DNA PRIMASE SMALL SUBUNIT / DNA PRIMASE 49 KDA SUBUNIT / P49


Mass: 50108.023 Da / Num. of mol.: 2 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): ROSETTA2
References: UniProt: P49642, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: Protein DNA POLYMERASE ALPHA CATALYTIC SUBUNIT, DNA PRIMASE LARGE SUBUNIT / DNA POLYMERASE ALPHA CATALYTIC SUBUNIT P180 / DNA PRIMASE 58 KDA SUBUNIT / P58


Mass: 30774.051 Da / Num. of mol.: 2
Fragment: PRIMASE-BINDING MOTIF RESIDUES 1445-1462, PRIL RESIDUES 19-253
Source method: isolated from a genetically manipulated source
Details: PRIL IS FUSED AT THE N-TERMINUS TO THE PRIMASE-BINDING MOTIF OF DNA POLYMERASE ALPHA (CHAIN E), VIA A 15-AMINO ACID LINKER OF SEQUENCE TGSTGSTGSTGSTGS
Source: (gene. exp.) HOMO SAPIENS (human) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): ROSETTA2
References: UniProt: P09884, UniProt: P49643, DNA-directed DNA polymerase, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
Sequence detailsCHAINS B AND D ARE ENGINEERED, CHIMERIC PROTEIN CONSTRUCTS. IN EACH CONSTRUCT, THE AMINO-TO- ...CHAINS B AND D ARE ENGINEERED, CHIMERIC PROTEIN CONSTRUCTS. IN EACH CONSTRUCT, THE AMINO-TO-CARBONYL ORDER IN THE CHAINS IS THE PRIMASE-BINDING MOTIF OF DNA POLYMERASE ALPHA AND THEN THE PRIL SUBUNIT OF PRIMASE. THE SUBUNITS ARE LINKED BY THE ARTIFICIAL TGSTGSTGSTGSTGS SEQUENCE, AND MAY APPEAR AS SEPARATE ENTITIES BECAUSE THE LINKER SEQUENCE IS NOT VISIBLE IN THE DENSITY MAP. CHAIN B: AMINO TERMINUS PRIMASE-BINDING MOTIF OF DNA POLYMERASE ALPHA IS NOT VISIBLE IN THE DENSITY MAP.

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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: 3.9 Å3/Da / Density % sol: 68 % / Description: NONE
Crystal growDetails: 100MM TRIS-HCL/BICINE PH 8.5, 20% GLYCEROL, 10% PEG4000 AND 20MM EACH OF AN ALCOHOL MIX COMPRISING 1,6-HEXANEDIOL, 1-BUTANOL, 1,2-PROPANEDIOL, 2-PROPANOL, 1,4- BUTANEDIOL AND 1,3-PROPANEDIOL.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 1.12713
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Sep 7, 2012
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.12713 Å / Relative weight: 1
ReflectionResolution: 3.4→49.37 Å / Num. obs: 28805 / % possible obs: 100 % / Observed criterion σ(I): 0 / Redundancy: 9.2 % / Biso Wilson estimate: 170.66 Å2 / Rmerge(I) obs: 0.06 / Net I/σ(I): 13.8
Reflection shellResolution: 3.4→3.58 Å / Redundancy: 9.4 % / Rmerge(I) obs: 1.5 / Mean I/σ(I) obs: 1.2 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 4BPU

4bpu


Resolution: 3.4→45.325 Å / SU ML: 0.63 / σ(F): 1.32 / Phase error: 34.39 / Stereochemistry target values: ML
Details: THE STRUCTURE WAS REFINED IN PHENIX WITH RIDING HYDROGENS. THE HYDROGENS HAVE BEEN INCLUDED IN THE ENTRY.
RfactorNum. reflection% reflection
Rfree0.2669 2864 5.2 %
Rwork0.2344 --
obs0.2361 28746 99.58 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 200.1 Å2
Refinement stepCycle: LAST / Resolution: 3.4→45.325 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9576 0 2 0 9578
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0049810
X-RAY DIFFRACTIONf_angle_d0.70313217
X-RAY DIFFRACTIONf_dihedral_angle_d11.2873728
X-RAY DIFFRACTIONf_chiral_restr0.0321422
X-RAY DIFFRACTIONf_plane_restr0.0041678
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.4-3.45860.54111380.46532513X-RAY DIFFRACTION95
3.4586-3.52150.40231360.39392653X-RAY DIFFRACTION99
3.5215-3.58920.39191220.34092636X-RAY DIFFRACTION100
3.5892-3.66240.3661370.31662657X-RAY DIFFRACTION100
3.6624-3.7420.40451560.30382633X-RAY DIFFRACTION100
3.742-3.8290.33931520.28282593X-RAY DIFFRACTION100
3.829-3.92470.32641480.2812661X-RAY DIFFRACTION100
3.9247-4.03080.3611830.27932537X-RAY DIFFRACTION100
4.0308-4.14930.34521300.25582689X-RAY DIFFRACTION100
4.1493-4.28310.3381330.25332611X-RAY DIFFRACTION100
4.2831-4.43610.25981410.21732644X-RAY DIFFRACTION100
4.4361-4.61360.25651420.20642598X-RAY DIFFRACTION100
4.6136-4.82330.23331160.1972708X-RAY DIFFRACTION100
4.8233-5.07730.22221380.20062641X-RAY DIFFRACTION100
5.0773-5.39490.24231410.20762659X-RAY DIFFRACTION100
5.3949-5.81070.26021590.22022610X-RAY DIFFRACTION100
5.8107-6.3940.29441670.23652618X-RAY DIFFRACTION100
6.394-7.31590.31381200.24742629X-RAY DIFFRACTION100
7.3159-9.20450.26671420.23192632X-RAY DIFFRACTION99
9.2045-45.32860.21641630.21882577X-RAY DIFFRACTION99

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