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- EMDB-40083: E. coli clamp loader with closed clamp on primed template DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-40083
TitleE. coli clamp loader with closed clamp on primed template DNA
Map dataCLC.DNA.Beta2 Closed
Sample
  • Complex: E. coli clamp loader with closed clamp on primed template DNA
    • Protein or peptide: x 5 types
    • DNA: x 2 types
  • Ligand: x 4 types
Keywordsclamp loader / DNA clamp / AAA+ / ATPase / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


DNA polymerase III, clamp loader complex / Hda-beta clamp complex / bacterial-type DNA replication / replication inhibiting complex / DNA polymerase III complex / replisome / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / DNA polymerase processivity factor activity / error-prone translesion synthesis ...DNA polymerase III, clamp loader complex / Hda-beta clamp complex / bacterial-type DNA replication / replication inhibiting complex / DNA polymerase III complex / replisome / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / DNA polymerase processivity factor activity / error-prone translesion synthesis / negative regulation of DNA-templated DNA replication initiation / 3'-5' exonuclease activity / ribonucleoside triphosphate phosphatase activity / response to radiation / DNA-templated DNA replication / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA damage response / ATP hydrolysis activity / protein homodimerization activity / DNA binding / ATP binding / identical protein binding / cytosol
Similarity search - Function
DNA polymerase III, psi subunit / DNA polymerase III, psi subunit, subgroup / DNA polymerase III, psi subunit superfamily / DNA polymerase III psi subunit / DNA polymerase III, delta prime subunit / DNA polymerase III, delta subunit, C-terminal / : / DNA polymerase III, delta subunit, C terminal / DNA polymerase III subunit delta', AAA+ ATPase lid domain / DNA polymerase III subunit delta, C-terminal ...DNA polymerase III, psi subunit / DNA polymerase III, psi subunit, subgroup / DNA polymerase III, psi subunit superfamily / DNA polymerase III psi subunit / DNA polymerase III, delta prime subunit / DNA polymerase III, delta subunit, C-terminal / : / DNA polymerase III, delta subunit, C terminal / DNA polymerase III subunit delta', AAA+ ATPase lid domain / DNA polymerase III subunit delta, C-terminal / Processivity clamp loader gamma complex DNA pol III C-term / DNA polymerase III, delta subunit / DNA polymerase III delta, N-terminal / DNA polymerase III, delta subunit / DNA polymerase III, tau subunit, domain V / DNA polymerase III subunit tau, DnaB-binding domain IV / DNA polymerase III, tau subunit, domain V superfamily / DNA polymerase III, subunit gamma/tau, helical lid domain / DNA polymerase III subunits tau domain IV DnaB-binding / DNA polymerase III tau subunit V interacting with alpha / DNA polymerase III, subunit gamma/ tau, N-terminal / DNA polymerase III, gamma subunit, domain III / DNA polymerase III subunits gamma and tau domain III / DNA polymerase III, delta subunit / DNA polymerase III, beta sliding clamp / DNA polymerase III, beta sliding clamp, N-terminal / DNA polymerase III, beta sliding clamp, C-terminal / DNA polymerase III, beta sliding clamp, central / DNA polymerase III beta subunit, N-terminal domain / DNA polymerase III beta subunit, central domain / DNA polymerase III beta subunit, C-terminal domain / DNA polymerase III beta subunit / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / ClpA/B family / : / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA polymerase III subunit tau / Beta sliding clamp / DNA polymerase III subunit delta / DNA polymerase III subunit delta' / DNA polymerase III subunit psi
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsOakley AJ / Xu Z-Q / Dixon NE
Funding support Australia, 1 items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP210100365 Australia
CitationJournal: To Be Published
Title: Structural characterisation of the complete cycle of sliding clamp loading in E. coli
Authors: Xu Z-Q / Oakley AJ / Dixon NE
History
DepositionMar 14, 2023-
Header (metadata) releaseMar 27, 2024-
Map releaseMar 27, 2024-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40083.png

Downloads & links

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Map

FileDownload / File: emd_40083.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCLC.DNA.Beta2 Closed
Voxel sizeX=Y=Z: 0.66 Å
Density
Contour LevelBy AUTHOR: 0.00463
Minimum - Maximum-0.0155699095 - 0.05136016
Average (Standard dev.)0.000005854174 (±0.0013632092)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 237.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: CLC.DNA.Beta2 Closed half map 2

Fileemd_40083_half_map_1.map
AnnotationCLC.DNA.Beta2 Closed half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CLC.DNA.Beta2 Closed half map 1

Fileemd_40083_half_map_2.map
AnnotationCLC.DNA.Beta2 Closed half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli clamp loader with closed clamp on primed template DNA

EntireName: E. coli clamp loader with closed clamp on primed template DNA
Components
  • Complex: E. coli clamp loader with closed clamp on primed template DNA
    • Protein or peptide: DNA polymerase III subunit deltaDNA polymerase III holoenzyme
    • Protein or peptide: DNA polymerase III subunit tauDNA polymerase III holoenzyme
    • Protein or peptide: DNA polymerase III subunit delta'DNA polymerase III holoenzyme
    • Protein or peptide: DNA polymerase III subunit psiDNA polymerase III holoenzyme
    • Protein or peptide: Beta sliding clamp
    • DNA: Primer
    • DNA: Template
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: ZINC ION

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Supramolecule #1: E. coli clamp loader with closed clamp on primed template DNA

SupramoleculeName: E. coli clamp loader with closed clamp on primed template DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Details: Clamp loader complex composed of DNA polymerase III delta tau(3) delta' chi and psi subunits. Clamp composed of DNA polymerase III beta(2) subunits.
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Molecular weightTheoretical: 419 KDa

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Macromolecule #1: DNA polymerase III subunit delta

MacromoleculeName: DNA polymerase III subunit delta / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 38.745574 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MIRLYPEQLR AQLNEGLRAA YLLLGNDPLL LQESQDAVRQ VAAAQGFEEH HTFSIDPNTD WNAIFSLCQA MSLFASRQTL LLLLPENGP NAAINEQLLT LTGLLHDDLL LIVRGNKLSK AQENAAWFTA LANRSVQVTC QTPEQAQLPR WVAARAKQLN L ELDDAANQ ...String:
MIRLYPEQLR AQLNEGLRAA YLLLGNDPLL LQESQDAVRQ VAAAQGFEEH HTFSIDPNTD WNAIFSLCQA MSLFASRQTL LLLLPENGP NAAINEQLLT LTGLLHDDLL LIVRGNKLSK AQENAAWFTA LANRSVQVTC QTPEQAQLPR WVAARAKQLN L ELDDAANQ VLCYCYEGNL LALAQALERL SLLWPDGKLT LPRVEQAVND AAHFTPFHWV DALLMGKSKR ALHILQQLRL EG SEPVILL RTLQRELLLL VNLKRQSAHT PLRALFDKHR VWQNRRGMMG EALNRLSQTQ LRQAVQLLTR TELTLKQDYG QSV WAELEG LSLLLCHKPL ADVFIDG

UniProtKB: DNA polymerase III subunit delta

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Macromolecule #2: DNA polymerase III subunit tau

MacromoleculeName: DNA polymerase III subunit tau / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 71.228648 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MSYQVLARKW RPQTFADVVG QEHVLTALAN GLSLGRIHHA YLFSGTRGVG KTSIARLLAK GLNCETGITA TPCGVCDNCR EIEQGRFVD LIEIDAASRT KVEDTRDLLD NVQYAPARGR FKVYLIDEVH MLSRHSFNAL LKTLEEPPEH VKFLLATTDP Q KLPVTILS ...String:
MSYQVLARKW RPQTFADVVG QEHVLTALAN GLSLGRIHHA YLFSGTRGVG KTSIARLLAK GLNCETGITA TPCGVCDNCR EIEQGRFVD LIEIDAASRT KVEDTRDLLD NVQYAPARGR FKVYLIDEVH MLSRHSFNAL LKTLEEPPEH VKFLLATTDP Q KLPVTILS RCLQFHLKAL DVEQIRHQLE HILNEEHIAH EPRALQLLAR AAEGSLRDAL SLTDQAIASG DGQVSTQAVS AM LGTLDDD QALSLVEAMV EANGERVMAL INEAAARGIE WEALLVEMLG LLHRIAMVQL SPAALGNDMA AIELRMRELA RTI PPTDIQ LYYQTLLIGR KELPYAPDRR MGVEMTLLRA LAFHPRMPLP EPEVPRQSFA PVAPTAVMTP TQVPPQPQSA PQQA PTVPL PETTSQVLAA RQQLQRVQGA TKAKKSEPAA ATRARPVNNA ALERLASVTD RVQARPVPSA LEKAPAKKEA YRWKA TTPV MQQKEVVATP KALKKALEHE KTPELAAKLA AEAIERDPWA AQVSQLSLPK LVEQVALNAW KEESDNAVCL HLRSSQ RHL NNRGAQQKLA EALSMLKGST VELTIVEDDN PAVRTPLEWR QAIYEEKLAQ ARESIIADNN IQTLRRFFDA ELDEESI RP I

UniProtKB: DNA polymerase III subunit tau

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Macromolecule #3: DNA polymerase III subunit delta'

MacromoleculeName: DNA polymerase III subunit delta' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 36.980484 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MRWYPWLRPD FEKLVASYQA GRGHHALLIQ ALPGMGDDAL IYALSRYLLC QQPQGHKSCG HCRGCQLMQA GTHPDYYTLA PEKGKNTLG VDAVREVTEK LNEHARLGGA KVVWVTDAAL LTDAAANALL KTLEEPPAET WFFLATREPE RLLATLRSRC R LHYLAPPP ...String:
MRWYPWLRPD FEKLVASYQA GRGHHALLIQ ALPGMGDDAL IYALSRYLLC QQPQGHKSCG HCRGCQLMQA GTHPDYYTLA PEKGKNTLG VDAVREVTEK LNEHARLGGA KVVWVTDAAL LTDAAANALL KTLEEPPAET WFFLATREPE RLLATLRSRC R LHYLAPPP EQYAVTWLSR EVTMSQDALL AALRLSAGSP GAALALFQGD NWQARETLCQ ALAYSVPSGD WYSLLAALNH EQ APARLHW LATLLMDALK RHHGAAQVTN VDVPGLVAEL ANHLSPSRLQ AILGDVCHIR EQLMSVTGIN RELLITDLLL RIE HYLQPG VVLPVPHL

UniProtKB: DNA polymerase III subunit delta'

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Macromolecule #4: DNA polymerase III subunit psi

MacromoleculeName: DNA polymerase III subunit psi / type: protein_or_peptide / ID: 4 / Details: DNA polymerase III subunit psi / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 15.188276 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MTSRRDWQLQ QLGITQWSLR RPGALQGEIA IAIPAHVRLV MVANDLPALT DPLVSDVLRA LTVSPDQVLQ LTPEKIAMLP QGSHCNSWR LGTDEPLSLE GAQVASPALT DLRANPTARA ALWQQICTYE HDFFPRND

UniProtKB: DNA polymerase III subunit psi

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Macromolecule #5: Beta sliding clamp

MacromoleculeName: Beta sliding clamp / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 40.630508 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MKFTVEREHL LKPLQQVSGP LGGRPTLPIL GNLLLQVADG TLSLTGTDLE MEMVARVALV QPHEPGATTV PARKFFDICR GLPEGAEIA VQLEGERMLV RSGRSRFSLS TLPAADFPNL DDWQSEVEFT LPQATMKRLI EATQFSMAHQ DVRYYLNGML F ETEGEELR ...String:
MKFTVEREHL LKPLQQVSGP LGGRPTLPIL GNLLLQVADG TLSLTGTDLE MEMVARVALV QPHEPGATTV PARKFFDICR GLPEGAEIA VQLEGERMLV RSGRSRFSLS TLPAADFPNL DDWQSEVEFT LPQATMKRLI EATQFSMAHQ DVRYYLNGML F ETEGEELR TVATDGHRLA VCSMPIGQSL PSHSVIVPRK GVIELMRMLD GGDNPLRVQI GSNNIRAHVG DFIFTSKLVD GR FPDYRRV LPKNPDKHLE AGCDLLKQAF ARAAILSNEK FRGVRLYVSE NQLKITANNP EQEEAEEILD VTYSGAEMEI GFN VSYVLD VLNALKCENV RMMLTDSVSS VQIEDAASQS AAYVVMPMRL

UniProtKB: Beta sliding clamp

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Macromolecule #6: Primer

MacromoleculeName: Primer / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 7.097624 KDa
SequenceString:
(DG)(DA)(DG)(DA)(DT)(DA)(DG)(DT)(DT)(DA) (DC)(DA)(DA)(DC)(DA)(DT)(DA)(DC)(DG)(DA) (DT)(DC)(DG)

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Macromolecule #7: Template

MacromoleculeName: Template / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 10.063474 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DC)(DG)(DA)(DT)(DC)(DG)(DT)(DA)(DT)(DG) (DT)(DT)(DG)(DT)(DA)(DA)(DC)(DT)(DA) (DT)(DC)(DT)(DC)

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Macromolecule #8: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 3 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 10 / Number of copies: 3 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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Macromolecule #11: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 11 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.5 mg/mL
BufferpH: 7.6
Component:
ConcentrationNameFormula
30.0 mMTris-HClTris
5.0 mMMagnesium ChlorideMgCl2
5.0 mMDithiothreitol
0.25 mMEDTAEthylenediaminetetraacetic acid
2.0 %Glycerol
2.0 mMAdenosine disphosphate
0.5 mMAluminium ChlorideAlCl3
5.0 mMSodium FluorideNaF

Details: 30 mM Tris-HCl pH 7.6, 5 mM MgCl2, 2 mM ADP, 0.5 mM AlCl3, 5 mM NaF, 5 mM dithiothreitol, 0.25 mM EDTA, 2% glycerol.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
Details: Used a Zepto Low-pressure plasma systems (PLASMA CLEANER) (Diener Electronic) at 10% power for 120 seconds.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV
Details: 3 uL of sample was applied onto a Ultrafoil Au R1.2/1.3 grid. Blot for 4.5 s with no extra force before plunging into liquid ethane..
Details30 microL of 6.0 microM delta/tau3/delta' complex was mixed with chi/psi complex, beta, and p/t DNA (annealed DNA oligonucleotides) at a molar ratio of 1:1.3 and dialysed at 4 degrees C against 250 mL of 30 mM Tris-HCl pH 7.6, 5 mM MgCl2, 2 mM ADP, 0.5 mM AlCl3, 5 mM NaF, 5 mM dithiothreitol, 0.25 mM EDTA, 2% glycerol.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.3 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Details: unfiltered mode
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 1 / Number real images: 1703 / Average exposure time: 6.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 600000
Startup modelType of model: OTHER / Details: Ab-initio model from RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 120000 / Software - Name: RELION (ver. 3.1)
Details: Most particles were classified to two classes that were used for final 3D reconstruction.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 452000

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Atomic model buiding 1

Initial model
PDB IDChain

3glh

source_name: PDB, initial_model_type: experimental model

1mmi

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8gj2:
E. coli clamp loader with closed clamp on primed template DNA

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