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- PDB-7b0y: Structure of a transcribing RNA polymerase II-U1 snRNP complex -

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Entry
Database: PDB / ID: 7b0y
TitleStructure of a transcribing RNA polymerase II-U1 snRNP complex
Components
  • (DNA-directed RNA polymerase II subunit ...Polymerase) x 8
  • (DNA-directed RNA polymerases I, II, and III subunit ...RNA polymerase) x 2
  • (Small nuclear ribonucleoprotein ...SnRNP) x 6
  • (U1 small nuclear ribonucleoprotein ...) x 2
  • 145-nt RNA
  • DNA-directed RNA polymerase subunit betaPolymerase
  • Non-template DNA
  • RPB12
  • Small nuclear ribonucleoprotein-associated protein
  • Template DNA
  • U1 snRNAU1 spliceosomal RNA
KeywordsSPLICING / Transcription / co-transcriptional spliceosome assembly
Function / homology
Function and homology information


negative regulation of protein refolding / U7 snRNA binding / histone pre-mRNA DCP binding / U7 snRNP / regulation of ATPase activity / regulation of mRNA polyadenylation / methylosome / histone mRNA metabolic process / negative regulation of chaperone-mediated autophagy / snRNA binding ...negative regulation of protein refolding / U7 snRNA binding / histone pre-mRNA DCP binding / U7 snRNP / regulation of ATPase activity / regulation of mRNA polyadenylation / methylosome / histone mRNA metabolic process / negative regulation of chaperone-mediated autophagy / snRNA binding / hair cycle / nuclear-transcribed mRNA catabolic process, exonucleolytic / telomerase holoenzyme complex / U1 snRNP binding / pICln-Sm protein complex / U4 snRNP / U2-type precatalytic spliceosome / positive regulation of mRNA splicing, via spliceosome / P granule / small nuclear ribonucleoprotein complex / spliceosomal tri-snRNP complex / telomerase RNA binding / recruitment of 3'-end processing factors to RNA polymerase II holoenzyme complex / SMN-Sm protein complex / import into nucleus / protein methylation / mRNA export from nucleus in response to heat stress / commitment complex / U2 snRNP / U2-type catalytic step 2 spliceosome / maintenance of transcriptional fidelity during DNA-templated transcription elongation from RNA polymerase II promoter / regulation of RNA splicing / U12-type spliceosomal complex / spliceosomal complex assembly / RNA polymerase II activity / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / U2-type prespliceosome / spliceosomal snRNP assembly / sno(s)RNA-containing ribonucleoprotein complex / U1 snRNP / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / precatalytic spliceosome / positive regulation of translational initiation / U5 snRNP / RNA polymerase I complex / mRNA cleavage / RNA polymerase III complex / RNA polymerase II, core complex / euchromatin => GO:0000791 / termination of RNA polymerase II transcription / U4/U6 x U5 tri-snRNP complex / U1 snRNA binding / translation initiation factor binding / core promoter sequence-specific DNA binding / catalytic step 2 spliceosome / spliceosomal complex / positive regulation of RNA splicing / RNA splicing / ribonucleoside binding / promoter-specific chromatin binding / P-body / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / microtubule cytoskeleton / transcription by RNA polymerase II / mRNA splicing, via spliceosome / transcription-coupled nucleotide-excision repair / single-stranded DNA binding / single-stranded RNA binding / ribonucleoprotein complex / nucleic acid binding / transcription initiation from RNA polymerase II promoter / nuclear body / nuclear speck / transcription, DNA-templated / protein dimerization activity / mRNA binding / nucleotide binding / nucleolus / enzyme binding / RNA binding / DNA binding / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 3 / RNA polymerase, N-terminal / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase Rpb4/RPC9, core ...DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 3 / RNA polymerase, N-terminal / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase Rpb4/RPC9, core / Archaeal RpoK/eukaryotic RPB6 RNA polymerase subunit / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase Rpb7-like , N-terminal / RNA polymerase subunit RPB4/RPC9 / RNA polymerase, Rpb8 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / S1 domain / Small nuclear ribonucleoprotein Sm D2 / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / Zinc finger, TFIIS-type / LSM domain, eukaryotic/archaea-type / RNA polymerase, subunit H/Rpb5 C-terminal / RNA polymerase, alpha subunit / RNA polymerase II, heptapeptide repeat, eukaryotic / RNA recognition motif domain / DNA-directed RNA polymerase, subunit N/Rpb10 / DNA-directed RNA polymerase, subunit RPB6 / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, conserved site / Small nuclear ribonucleoprotein G / Nucleotide-binding alpha-beta plait domain superfamily / RNA polymerase subunit RPB10 / RNA-binding domain, S1 / U1 small nuclear ribonucleoprotein of 70kDa N-terminal / RNA polymerases, subunit N, zinc binding site / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / Archaeal RpoH /eukaryotic RPB5 RNA polymerase subunit / RNA polymerase, subunit H/Rpb5, conserved site / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / Small ribonucleoprotein associated, SmB/SmN / Sm-like protein Lsm6/SmF / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase RPB5 subunit, eukaryote/virus / Nucleic acid-binding, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase subunit/transcription factor S / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain / HRDC-like superfamily / LSM domain superfamily / DNA-directed RNA polymerase, RBP11-like dimerisation domain / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / Pol II subunit B9, C-terminal zinc ribbon / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein Sm D3 / RNA polymerase RBP11 / Rpb4/RPC9 superfamily / Small nuclear ribonucleoprotein F / Small nuclear ribonucleoprotein D1 / snRNP70, RNA recognition motif / U1 small nuclear ribonucleoprotein A, RNA recognition motif 1 / RNA polymerase Rpb1, funnel domain superfamily / U1 small nuclear ribonucleoprotein A, RNA recognition motif 2 / RPB5-like RNA polymerase subunit superfamily / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA-binding domain superfamily / RPB6/omega subunit-like superfamily / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase Rpb1, domain 7 superfamily / DNA-directed RNA polymerase subunit Rpb5-like / RNA polymerase Rpb1, domain 3 superfamily
Small nuclear ribonucleoprotein F / DNA-directed RNA polymerase II subunit RPB9 / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein Sm D1 / Small nuclear ribonucleoprotein Sm D2 / U1 small nuclear ribonucleoprotein A / DNA-directed RNA polymerase II subunit RPB1 / DNA-directed RNA polymerase II subunit RPB3 ...Small nuclear ribonucleoprotein F / DNA-directed RNA polymerase II subunit RPB9 / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein Sm D1 / Small nuclear ribonucleoprotein Sm D2 / U1 small nuclear ribonucleoprotein A / DNA-directed RNA polymerase II subunit RPB1 / DNA-directed RNA polymerase II subunit RPB3 / U1 small nuclear ribonucleoprotein 70 kDa / DNA-directed RNA polymerase II subunit E / RNA polymerase II subunit K / DNA-directed RNA polymerase II subunit RPB7 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase II subunit F / DNA-directed RNA polymerase II subunit RPB11-a / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / RNA polymerase II subunit D / DNA-directed RNA polymerase subunit beta / Small nuclear ribonucleoprotein-associated protein
Biological speciesSus scrofa domesticus (domestic pig)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsZhang, S. / Aibara, S. / Vos, S.M. / Agafonov, D.E. / Luehrmann, R. / Cramer, P.
Funding support4items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)ALTF 830-2018
H2020 Marie Curie Actions of the European Commission894862
European Research Council (ERC)882357
German Research Foundation (DFG)EXC 2067/1-390729940
CitationJournal: Science / Year: 2021
Title: Structure of a transcribing RNA polymerase II-U1 snRNP complex.
Authors: Suyang Zhang / Shintaro Aibara / Seychelle M Vos / Dmitry E Agafonov / Reinhard Lührmann / Patrick Cramer /
Abstract: To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA (pre-mRNA). Here, we report ...To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA (pre-mRNA). Here, we report the cryo-electron microscopy structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the pre-mRNA 5' splice site near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5' splice site, leading to the formation of a "growing intron loop." Loop formation may facilitate scanning of nascent pre-mRNA for the 3' splice site, functional pairing of distant intron ends, and prespliceosome assembly. Our results provide a starting point for a mechanistic analysis of cotranscriptional spliceosome assembly and the biogenesis of mRNA isoforms by alternative splicing.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 23, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 27, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase II subunit RPB1
B: DNA-directed RNA polymerase subunit beta
C: DNA-directed RNA polymerase II subunit RPB3
D: DNA-directed RNA polymerase II subunit D
E: DNA-directed RNA polymerase II subunit E
F: DNA-directed RNA polymerase II subunit F
G: DNA-directed RNA polymerase II subunit RPB7
H: DNA-directed RNA polymerases I, II, and III subunit RPABC3
I: DNA-directed RNA polymerase II subunit RPB9
J: DNA-directed RNA polymerases I, II, and III subunit RPABC5
K: DNA-directed RNA polymerase II subunit RPB11-a
L: RPB12
N: Non-template DNA
P: 145-nt RNA
T: Template DNA
a: U1 snRNA
b: U1 small nuclear ribonucleoprotein 70 kDa
c: U1 small nuclear ribonucleoprotein A
e: Small nuclear ribonucleoprotein Sm D2
f: Small nuclear ribonucleoprotein F
g: Small nuclear ribonucleoprotein E
h: Small nuclear ribonucleoprotein G
i: Small nuclear ribonucleoprotein Sm D3
j: Small nuclear ribonucleoprotein-associated protein
k: Small nuclear ribonucleoprotein Sm D1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)823,57034
Polymers823,02325
Non-polymers5489
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area106390 Å2
ΔGint-570 kcal/mol
Surface area223810 Å2
MethodPISA

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Components

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DNA-directed RNA polymerase II subunit ... , 8 types, 8 molecules ACDEFGIK

#1: Protein DNA-directed RNA polymerase II subunit RPB1 / Polymerase / RNA polymerase II subunit B1 / DNA-directed RNA polymerase II subunit A / DNA-directed RNA ...RNA polymerase II subunit B1 / DNA-directed RNA polymerase II subunit A / DNA-directed RNA polymerase III largest subunit


Mass: 217450.078 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: P11414, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase II subunit RPB3 / Polymerase


Mass: 31439.074 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: I3LCH3
#4: Protein DNA-directed RNA polymerase II subunit D / Polymerase


Mass: 16331.255 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: A0A287ADR4
#5: Protein DNA-directed RNA polymerase II subunit E / Polymerase / RPB5 homolog


Mass: 24644.318 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: I3LSI7
#6: Protein DNA-directed RNA polymerase II subunit F / Polymerase / DNA-directed RNA polymerases I / II / and III subunit RPABC2 / RPB6 homolog


Mass: 14477.001 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: F1SKN8
#7: Protein DNA-directed RNA polymerase II subunit RPB7 / Polymerase


Mass: 19314.283 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: I3LJZ9
#9: Protein DNA-directed RNA polymerase II subunit RPB9 / Polymerase / RNA polymerase II subunit B9 / DNA-directed RNA polymerase II subunit I / RNA polymerase II 14.5 ...RNA polymerase II subunit B9 / DNA-directed RNA polymerase II subunit I / RNA polymerase II 14.5 kDa subunit / RPB14.5


Mass: 14541.221 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: P60899
#11: Protein DNA-directed RNA polymerase II subunit RPB11-a / Polymerase


Mass: 13310.284 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: F1RKE4

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Protein , 3 types, 3 molecules BLj

#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase


Mass: 134041.422 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig)
References: UniProt: A0A0B8RVL1, DNA-directed RNA polymerase
#12: Protein RPB12


Mass: 7018.244 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: I3LN51
#24: Protein Small nuclear ribonucleoprotein-associated protein


Mass: 23686.004 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q66K91

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DNA-directed RNA polymerases I, II, and III subunit ... , 2 types, 2 molecules HJ

#8: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC3 / RNA polymerase


Mass: 17162.273 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: I3LCB2
#10: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC5 / RNA polymerase


Mass: 7655.123 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: A0A4X1VYD0

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DNA chain , 2 types, 2 molecules NT

#13: DNA chain Non-template DNA


Mass: 14932.533 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#15: DNA chain Template DNA


Mass: 14672.335 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain , 2 types, 2 molecules Pa

#14: RNA chain 145-nt RNA


Mass: 46784.910 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#16: RNA chain U1 snRNA / U1 spliceosomal RNA


Mass: 52697.070 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)

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U1 small nuclear ribonucleoprotein ... , 2 types, 2 molecules bc

#17: Protein U1 small nuclear ribonucleoprotein 70 kDa / snRNP70


Mass: 51683.184 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P08621
#18: Protein U1 small nuclear ribonucleoprotein A / U1A


Mass: 31319.533 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P09012

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Small nuclear ribonucleoprotein ... , 6 types, 6 molecules efghik

#19: Protein Small nuclear ribonucleoprotein Sm D2 / Sm-D2 / snRNP core protein D2


Mass: 13551.928 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62316
#20: Protein Small nuclear ribonucleoprotein F / snRNP-F / Sm protein F / SmF


Mass: 9734.171 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62306
#21: Protein Small nuclear ribonucleoprotein E / snRNP-E / Sm protein E / SmE


Mass: 10817.601 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62304
#22: Protein Small nuclear ribonucleoprotein G / snRNP-G / Sm protein G / SmG


Mass: 8508.084 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62308
#23: Protein Small nuclear ribonucleoprotein Sm D3 / Sm-D3 / snRNP core protein D3


Mass: 13940.308 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62318
#25: Protein Small nuclear ribonucleoprotein Sm D1 / Sm-D1 / Sm-D autoantigen / snRNP core protein D1


Mass: 13310.653 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62314

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Non-polymers , 2 types, 9 molecules

#26: Chemical
ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn
#27: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1The transcribing RNA polymerase II-U1 snRNP complex with 145-nt RNACOMPLEX#1-#250MULTIPLE SOURCES
2RNA polymerase IICOMPLEX#1-#121NATURAL
3Nucleic acidsNucleic acidCOMPLEX#13-#151RECOMBINANT
4U1 snRNPCOMPLEX#16-#251NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Sus scrofa domesticus (domestic pig)9825
23synthetic construct (others)32630
34Homo sapiens (human)9606
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.5
Buffer component
IDConc.FormulaBuffer-ID
120 mMHEPES1
2150 mMNaClSodium chloride1
33 mMMgCl21
40.5 mMTCEP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 1.01 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1Warpparticle selection
2SerialEMimage acquisition
4WarpCTF correction
5RELIONCTF correction
8Cootmodel fitting
9UCSF Chimeramodel fitting
11PHENIXmodel refinement
15RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61596 / Symmetry type: POINT
Atomic model building
IDPDB-ID3D fitting-ID
16GMH1
23PGW1
34PJO1
44PKD1

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