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- EMDB-27099: Human CST-DNA polymerase alpha/primase preinitiation complex boun... -

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Basic information

Entry
Database: EMDB / ID: EMD-27099
TitleHuman CST-DNA polymerase alpha/primase preinitiation complex bound to 3xTEL template - local refined domains and merged
Map dataMerged map from three local refined domains.
Sample
  • Complex: Human CST-DNA polymerase alpha/primase preinitiation complex bound to 3xTEL template - local refined domains and merged
    • Protein or peptide: CTC1
    • Protein or peptide: STN1
    • Protein or peptide: TEN1
    • Protein or peptide: PRIM1
    • Protein or peptide: PRIM2
    • Protein or peptide: POLA1
    • Protein or peptide: POLA2
    • DNA: C-3xTEL
Keywordstelomere / C-strand / preinitiation complex / 3xTEL template / local refinement / merged / REPLICATION-DNA complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.61 Å
AuthorsHe Q / Lim C
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM131023 United States
CitationJournal: Nature / Year: 2022
Title: Structures of the human CST-Polα-primase complex bound to telomere templates.
Authors: Qixiang He / Xiuhua Lin / Bianca L Chavez / Sourav Agrawal / Benjamin L Lusk / Ci Ji Lim /
Abstract: The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways such as lagging-strand ...The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study.
History
DepositionMay 24, 2022-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27099.png

Downloads & links

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Map

FileDownload / File: emd_27099.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMerged map from three local refined domains.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 400 pix.
= 432. Å		1.08 Å/pix.
x 400 pix.
= 432. Å		1.08 Å/pix.
x 400 pix.
= 432. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-0.0383943 - 3.2036119
Average (Standard dev.)0.004585028 (±0.052227154)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Human CST-DNA polymerase alpha/primase preinitiation complex boun...

EntireName: Human CST-DNA polymerase alpha/primase preinitiation complex bound to 3xTEL template - local refined domains and merged
Components
  • Complex: Human CST-DNA polymerase alpha/primase preinitiation complex bound to 3xTEL template - local refined domains and merged
    • Protein or peptide: CTC1
    • Protein or peptide: STN1
    • Protein or peptide: TEN1
    • Protein or peptide: PRIM1
    • Protein or peptide: PRIM2
    • Protein or peptide: POLA1
    • Protein or peptide: POLA2
    • DNA: C-3xTEL

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Supramolecule #1: Human CST-DNA polymerase alpha/primase preinitiation complex boun...

SupramoleculeName: Human CST-DNA polymerase alpha/primase preinitiation complex bound to 3xTEL template - local refined domains and merged
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 540 KDa

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Macromolecule #1: CTC1

MacromoleculeName: CTC1 / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGDYKDHDGD YKDHDIDYKD DDDKSGGSLE AAGRAQVPSS EQAWLEDAQV FIQKTLCPAV KEPNVQLTPL VIDCVKTVWL SQGRNQGSTL PLSYSFVSVQ DLKTHQRLPC CSHLSWSSSA YQAWAQEAGP NGNPLPREQL LLLGTLTDLS ADLEQECRNG SLYVRDNTGV ...String:
MGDYKDHDGD YKDHDIDYKD DDDKSGGSLE AAGRAQVPSS EQAWLEDAQV FIQKTLCPAV KEPNVQLTPL VIDCVKTVWL SQGRNQGSTL PLSYSFVSVQ DLKTHQRLPC CSHLSWSSSA YQAWAQEAGP NGNPLPREQL LLLGTLTDLS ADLEQECRNG SLYVRDNTGV LSCELIDLDL SWLGHLFLFP RWSYLPPARW NSSGEGHLEL WDAPVPVFPL TISPGPVTPI PVLYPESASC LLRLRNKLRG VQRNLAGSLV RLSALVKSKQ KAYFILSLGR SHPAVTHVSI IVQVPAQLVW HRALRPGTAY VLTELRVSKI RGQRQHVWMT SQSSRLLLLK PECVQELELE LEGPLLEADP KPLPMPSNSE DKKDPESLVR YSRLLSYSGA VTGVLNEPAG LYELDGQLGL CLAYQQFRGL RRVMRPGVCL QLQDVHLLQS VGGGTRRPVL APCLRGAVLL QSFSRQKPGA HSSRQAYGAS LYEQLVWERQ LGLPLYLWAT KALEELACKL CPHVLRHHQF LQHSSPGSPS LGLQLLAPTL DLLAPPGSPV RNAHNEILEE PHHCPLQKYT RLQTPSSFPT LATLKEEGQR KAWASFDPKA LLPLPEASYL PSCQLNRRLA WSWLCLLPSA FCPAQVLLGV LVASSHKGCL QLRDQSGSLP CLLLAKHSQP LSDPRLIGCL VRAERFQLIV ERDVRSSFPS WKELSMPGFI QKQQARVYVQ FFLADALILP VPRPCLHSAT PSTPQTDPTG PEGPHLGQSR LFLLCHKEAL MKRNFCVPPG ASPEVPKPAL SFYVLGSWLG GTQRKEGTGW GLPEPQGNDD NDQKVHLIFF GSSVRWFEFL HPGQVYRLVA PGPATPMLFE KDGSSCISRR PLELAGCASC LTVQDNWTLE LESSQDIQDV LDANKSLPES SLTDLLSDNF TDSLVSFSAE ILSRTLCEPL VASLWMKLGN TGAMRRCVKL TVALETAECE FPPHLDVYIE DPHLPPSLGL LPGARVHFSQ LEKRVSRSHN VYCCFRSSTY VQVLSFPPET TISVPLPHIY LAELLQGGQS PFQATASCHI VSVFSLQLFW VCAYCTSICR QGKCTRLGST CPTQTAISQA IIRLLVEDGT AEAVVTCRNH HVAAALGLCP REWASLLDFV QVPGRVVLQF AGPGAQLESS ARVDEPMTMF LWTLCTSPSV LRPIVLSFEL ERKPSKIVPL EPPRLQRFQC GELPFLTHVN PRLRLSCLSI RESEYSSSLG ILASSC

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Macromolecule #2: STN1

MacromoleculeName: STN1 / type: protein_or_peptide / ID: 2 / Enantiomer: DEXTRO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MHHHHHHQPG SSRCEEETPS LLWGLDPVFL AFAKLYIRDI LDMKESRQVP GVFLYNGHPI KQVDVLGTVI GVRERDAFYS YGVDDSTGVI NCICWKKLNT ESVSAAPSAA RELSLTSQLK KLQETIEQKT KIEIGDTIRV RGSIRTYREE REIHATTYYK VDDPVWNIQI ...String:
MHHHHHHQPG SSRCEEETPS LLWGLDPVFL AFAKLYIRDI LDMKESRQVP GVFLYNGHPI KQVDVLGTVI GVRERDAFYS YGVDDSTGVI NCICWKKLNT ESVSAAPSAA RELSLTSQLK KLQETIEQKT KIEIGDTIRV RGSIRTYREE REIHATTYYK VDDPVWNIQI ARMLELPTIY RKVYDQPFHS SALEKEEALS NPGALDLPSL TSLLSEKAKE FLMENRVQSF YQQELEMVES LLSLANQPVI HSASSDQVNF KKDTTSKAIH SIFKNAIQLL QEKGLVFQKD DGFDNLYYVT REDKDLHRKI HRIIQQDCQK PNHMEKGCHF LHILACARLS IRPGLSEAVL QQVLELLEDQ SDIVSTMEHY YTAF

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Macromolecule #3: TEN1

MacromoleculeName: TEN1 / type: protein_or_peptide / ID: 3 / Enantiomer: DEXTRO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MSYYHHHHHH DYDIPTTENL YFQGAMGSGI QLPKPGTYYL PWEVSAGQVP DGSTLRTFGR LCLYDMIQSR VTLMAQHGSD QHQVLVCTKL VEPFHAQVGS LYIVLGELQH QQDRGSVVKA RVLTCVEGMN LPLLEQAIRE QRLYKQERGG SQ

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Macromolecule #4: PRIM1

MacromoleculeName: PRIM1 / type: protein_or_peptide / ID: 4 / Enantiomer: DEXTRO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGHHHHHHGS GSGSGETFDP TELPELLKLY YRRLFPYSQY YRWLNYGGVI KNYFQHREFS FTLKDDIYIR YQSFNNQSDL EKEMQKMNPY KIDIGAVYSH RPNQHNTVKL GAFQAQEKEL VFDIDMTDYD DVRRCCSSAD ICPKCWTLMT MAIRIIDRAL KEDFGFKHRL ...String:
MGHHHHHHGS GSGSGETFDP TELPELLKLY YRRLFPYSQY YRWLNYGGVI KNYFQHREFS FTLKDDIYIR YQSFNNQSDL EKEMQKMNPY KIDIGAVYSH RPNQHNTVKL GAFQAQEKEL VFDIDMTDYD DVRRCCSSAD ICPKCWTLMT MAIRIIDRAL KEDFGFKHRL WVYSGRRGVH CWVCDESVRK LSSAVRSGIV EYLSLVKGGQ DVKKKVHLSE KIHPFIRKSI NIIKKYFEEY ALVNQDILEN KESWDKILAL VPETIHDELQ QSFQKSHNSL QRWEHLKKVA SRYQNNIKND KYGPWLEWEI MLQYCFPRLD INVSKGINHL LKSPFSVHPK TGRISVPIDL QKVDQFDPFT VPTISFICRE LDAISTNEEE KEENEAESDV KHRTRDYKKT SLAPYVKVFE HFLENLDKSR KGELLKKSDL QKDF

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Macromolecule #5: PRIM2

MacromoleculeName: PRIM2 / type: protein_or_peptide / ID: 5 / Enantiomer: DEXTRO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGHHHHHHGS GSGSGEFSGR KWRKLRLAGD QRNASYPHCL QFYLQPPSEN ISLIEFENLA IDRVKLLKSV ENLGVSYVKG TEQYQSKLES ELRKLKFSYR ENLEDEYEPR RRDHISHFIL RLAYCQSEEL RRWFIQQEMD LLRFRFSILP KDKIQDFLKD SQLQFEAISD ...String:
MGHHHHHHGS GSGSGEFSGR KWRKLRLAGD QRNASYPHCL QFYLQPPSEN ISLIEFENLA IDRVKLLKSV ENLGVSYVKG TEQYQSKLES ELRKLKFSYR ENLEDEYEPR RRDHISHFIL RLAYCQSEEL RRWFIQQEMD LLRFRFSILP KDKIQDFLKD SQLQFEAISD EEKTLREQEI VASSPSLSGL KLGFESIYKI PFADALDLFR GRKVYLEDGF AYVPLKDIVA IILNEFRAKL SKALALTARS LPAVQSDERL QPLLNHLSHS YTGQDYSTQG NVGKISLDQI DLLSTKSFPP CMRQLHKALR ENHHLRHGGR MQYGLFLKGI GLTLEQALQF WKQEFIKGKM DPDKFDKGYS YNIRHSFGKE GKRTDYTPFS CLKIILSNPP SQGDYHGCPF RHSDPELLKQ KLQSYKISPG GISQILDLVK GTHYQVACQK YFEMIHNVDD CGFSLNHPNQ FFCESQRILN GGKDIKKEPI QPETPQPKPS VQKTKDASSA LASLNSSLEM DMEGLEDYFS EDS

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Macromolecule #6: POLA1

MacromoleculeName: POLA1 / type: protein_or_peptide / ID: 6 / Enantiomer: DEXTRO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGGSAGDYKD HDGDYKDHDI DYKDDDDKGA SSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KGAGSAPVHG DDSLSDSGSF VSSRARREKK SKKGRQEALE RLKKAKAGEK YKYEVEDFTG VYEEVDEEQY SKLVQARQDD DWIVDDDGIG YVEDGREIFD DDLEDDALDA ...String:
MGGSAGDYKD HDGDYKDHDI DYKDDDDKGA SSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KGAGSAPVHG DDSLSDSGSF VSSRARREKK SKKGRQEALE RLKKAKAGEK YKYEVEDFTG VYEEVDEEQY SKLVQARQDD DWIVDDDGIG YVEDGREIFD DDLEDDALDA DEKGKDGKAR NKDKRNVKKL AVTKPNNIKS MFIACAGKKT ADKAVDLSKD GLLGDILQDL NTETPQITPP PVMILKKKRS IGASPNPFSV HTATAVPSGK IASPVSRKEP PLTPVPLKRA EFAGDDVQVE STEEEQESGA MEFEDGDFDE PMEVEEVDLE PMAAKAWDKE SEPAEEVKQE ADSGKGTVSY LGSFLPDVSC WDIDQEGDSS FSVQEVQVDS SHLPLVKGAD EEQVFHFYWL DAYEDQYNQP GVVFLFGKVW IESAETHVSC CVMVKNIERT LYFLPREMKI DLNTGKETGT PISMKDVYEE FDEKIATKYK IMKFKSKPVE KNYAFEIPDV PEKSEYLEVK YSAEMPQLPQ DLKGETFSHV FGTNTSSLEL FLMNRKIKGP CWLEVKSPQL LNQPVSWCKV EAMALKPDLV NVIKDVSPPP LVVMAFSMKT MQNAKNHQNE IIAMAALVHH SFALDKAAPK PPFQSHFCVV SKPKDCIFPY AFKEVIEKKN VKVEVAATER TLLGFFLAKV HKIDPDIIVG HNIYGFELEV LLQRINVCKA PHWSKIGRLK RSNMPKLGGR SGFGERNATC GRMICDVEIS AKELIRCKSY HLSELVQQIL KTERVVIPME NIQNMYSESS QLLYLLEHTW KDAKFILQIM CELNVLPLAL QITNIAGNIM SRTLMGGRSE RNEFLLLHAF YENNYIVPDK QIFRKPQQKL GDEDEEIDGD TNKYKKGRKK AAYAGGLVLD PKVGFYDKFI LLLDFNSLYP SIIQEFNICF TTVQRVASEA QKVTEDGEQE QIPELPDPSL EMGILPREIR KLVERRKQVK QLMKQQDLNP DLILQYDIRQ KALKLTANSM YGCLGFSYSR FYAKPLAALV TYKGREILMH TKEMVQKMNL EVIYGDTDSI MINTNSTNLE EVFKLGNKVK SEVNKLYKLL EIDIDGVFKS LLLLKKKKYA ALVVEPTSDG NYVTKQELKG LDIVRRDWCD LAKDTGNFVI GQILSDQSRD TIVENIQKRL IEIGENVLNG SVPVSQFEIN KALTKDPQDY PDKKSLPHVH VALWINSQGG RKVKAGDTVS YVICQDGSNL TASQRAYAPE QLQKQDNLTI DTQYYLAQQI HPVVARICEP IDGIDAVLIA TWLGLDPTQF RVHHYHKDEE NDALLGGPAQ LTDEEKYRDC ERFKCPCPTC GTENIYDNVF DGSGTDMEPS LYRCSNIDCK ASPLTFTVQL SNKLIMDIRR FIKKYYDGWL ICEEPTCRNR TRHLPLQFSR TGPLCPACMK ATLQPEYSDK SLYTQLCFYR YIFDAECALE KLTTDHEKDK LKKQFFTPKV LQDYRKLKNT AEQFLSRSGY SEVNLSKLFA GCAVKSV

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Macromolecule #7: POLA2

MacromoleculeName: POLA2 / type: protein_or_peptide / ID: 7 / Enantiomer: DEXTRO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGHHHHHHGS GSGSGSASAQ QLAEELQIFG LDCEEALIEK LVELCVQYGQ NEEGMVGELI AFCTSTHKVG LTSEILNSFE HEFLSKRLSK ARHSTCKDSG HAGARDIVSI QELIEVEEEE EILLNSYTTP SKGSQKRAIS TPETPLTKRS VSTRSPHQLL SPSSFSPSAT ...String:
MGHHHHHHGS GSGSGSASAQ QLAEELQIFG LDCEEALIEK LVELCVQYGQ NEEGMVGELI AFCTSTHKVG LTSEILNSFE HEFLSKRLSK ARHSTCKDSG HAGARDIVSI QELIEVEEEE EILLNSYTTP SKGSQKRAIS TPETPLTKRS VSTRSPHQLL SPSSFSPSAT PSQKYNSRSN RGEVVTSFGL AQGVSWSGRG GAGNISLKVL GCPEALTGSY KSMFQKLPDI REVLTCKIEE LGSELKEHYK IEAFTPLLAP AQEPVTLLGQ IGCDSNGKLN NKSVILEGDR EHSSGAQIPV DLSELKEYSL FPGQVVIMEG INTTGRKLVA TKLYEGVPLP FYQPTEEDAD FEQSMVLVAC GPYTTSDSIT YDPLLDLIAV INHDRPDVCI LFGPFLDAKH EQVENCLLTS PFEDIFKQCL RTIIEGTRSS GSHLVFVPSL RDVHHEPVYP QPPFSYSDLS REDKKQVQFV SEPCSLSING VIFGLTSTDL LFHLGAEEIS SSSGTSDRFS RILKHILTQR SYYPLYPPQE DMAIDYESFY VYAQLPVTPD VLIIPSELRY FVKDVLGCVC VNPGRLTKGQ VGGTFARLYL RRPAADGAER QSPCIAVQVV RI

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Macromolecule #8: C-3xTEL

MacromoleculeName: C-3xTEL / type: dna / ID: 8 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
CTTAGGGTTA GGGTTAGGG

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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

Concentration5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMHEPES-NaHEPES
150.0 mMNaClSodium Chloride
2.0 mMMgCl2Magnesium Chloride
1.0 mMTCEPTCEP
4.0 mMCHAPSOCHAPSO
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Details: 15 mA using PELCO EasiGLOW
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 6251 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 222188
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.61 Å / Resolution method: OTHER / Software - Name: cryoSPARC
Details: Three local refined domains merged using vop maximum command in Chimera
Number images used: 164324
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION

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