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- PDB-6exv: Structure of mammalian RNA polymerase II elongation complex inhib... -

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Entry
Database: PDB / ID: 6exv
TitleStructure of mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitin
Components
  • (DNA-DIRECTED RNA POLYMERASE II SUBUNIT ...) x 6
  • (DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT ...) x 6
  • AMATOXIN
  • DNA (25-MER)
  • DNA (36-MER)
  • RNA (5'-R(P*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')
KeywordsTRANSCRIPTION / Inhibitor / elongation / active site
Function / homology
Function and homology information


: / Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / Transcriptional regulation by small RNAs / RNA Polymerase II Pre-transcription Events / TP53 Regulates Transcription of DNA Repair Genes / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / mRNA Capping / mRNA Splicing - Minor Pathway ...: / Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / Transcriptional regulation by small RNAs / RNA Polymerase II Pre-transcription Events / TP53 Regulates Transcription of DNA Repair Genes / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / mRNA Capping / mRNA Splicing - Minor Pathway / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Elongation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Pol II CTD phosphorylation and interaction with CE / Estrogen-dependent gene expression / Formation of TC-NER Pre-Incision Complex / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / mRNA Splicing - Major Pathway / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / organelle membrane / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / positive regulation of translational initiation / RNA polymerase I complex / transcription elongation by RNA polymerase I / RNA polymerase III complex / core promoter sequence-specific DNA binding / RNA polymerase II, core complex / tRNA transcription by RNA polymerase III / transcription by RNA polymerase I / translation initiation factor binding / transcription-coupled nucleotide-excision repair / transcription initiation at RNA polymerase II promoter / P-body / euchromatin / ribonucleoside binding / fibrillar center / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / single-stranded DNA binding / toxin activity / nucleic acid binding / transcription by RNA polymerase II / chromosome, telomeric region / single-stranded RNA binding / protein dimerization activity / nuclear speck / nucleotide binding / RNA-directed RNA polymerase activity / DNA-templated transcription / chromatin binding / nucleolus / mitochondrion / DNA binding / zinc ion binding / metal ion binding / nucleus / cytosol
Similarity search - Function
RNA Polymerase II, Rpb4 subunit / RNA polymerase Rpb7-like, N-terminal domain / Dna-directed Rna Polymerases I, Ii, And Iii 27 Kd Polypeptide; Chain: A; domain 1 / RNA polymerase, Rpb5, N-terminal domain / RNA polymerase ii, chain L / Gyrase A; domain 2 - #140 / RPB5-like RNA polymerase subunit / RNA-binding domain, S1 / N-terminal domain of TfIIb - #10 / Dna-directed Rna Polymerase Ii 140kd Polypeptide; Chain: B; domain 3 ...RNA Polymerase II, Rpb4 subunit / RNA polymerase Rpb7-like, N-terminal domain / Dna-directed Rna Polymerases I, Ii, And Iii 27 Kd Polypeptide; Chain: A; domain 1 / RNA polymerase, Rpb5, N-terminal domain / RNA polymerase ii, chain L / Gyrase A; domain 2 - #140 / RPB5-like RNA polymerase subunit / RNA-binding domain, S1 / N-terminal domain of TfIIb - #10 / Dna-directed Rna Polymerase Ii 140kd Polypeptide; Chain: B; domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / RNA polymerase Rpb1, domain 3 / RNA polymerase, RBP11-like subunit / Enzyme I; Chain A, domain 2 / N-terminal domain of TfIIb / Rubrerythrin, domain 2 / Growth Hormone; Chain: A; / DNA-directed RNA polymerase, insert domain / RNA Polymerase Alpha Subunit; Chain A, domain 2 / DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb1 C-terminal repeat / RNA polymerase II, heptapeptide repeat, eukaryotic / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / Gyrase A; domain 2 / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / Rpb4/RPC9 superfamily / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / Homeodomain-like / Zinc finger TFIIS-type signature. / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase subunit Rpb7-like / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / HRDC-like superfamily / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / : / RNA polymerase, Rpb8 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerase Rpb8 / RNA polymerase subunit 8 / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb5, N-terminal domain / DNA-directed RNA polymerase, subunit RPB6 / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerases L / 13 to 16 Kd subunits signature. / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / Single Sheet / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / Nucleic acid-binding proteins / Dna Ligase; domain 1 / S1 domain / Beta Complex / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6
Similarity search - Domain/homology
Alpha-Amanitin / DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase II subunit RPB4 / DNA-directed RNA polymerase II subunit RPB11-a / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase II subunit RPB3 ...Alpha-Amanitin / DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase II subunit RPB4 / DNA-directed RNA polymerase II subunit RPB11-a / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase II subunit RPB7 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerase II subunit RPB9 / Alpha-amanitin proprotein
Similarity search - Component
Biological speciesSus scrofa (pig)
Sus Scrofa (pig)
Amanita phalloides (death cap)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsLiu, X. / Farnung, L. / Wigge, C. / Cramer, P.
Funding support Germany, 3items
OrganizationGrant numberCountry
European Research Council693023 Germany
German Research FoundationSFB860 Germany
German Research FoundationSPP1935 Germany
CitationJournal: J Biol Chem / Year: 2018
Title: Cryo-EM structure of a mammalian RNA polymerase II elongation complex inhibited by α-amanitin.
Authors: Xiangyang Liu / Lucas Farnung / Christoph Wigge / Patrick Cramer /
Abstract: RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step ...RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II but forms additional contacts with metazoan-specific residues, which explains why its affinity to mammalian Pol II is ∼3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.
History
DepositionNov 9, 2017Deposition site: PDBE / Processing site: PDBE
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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 II SUBUNIT RPB2
C: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3
D: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3
E: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1
F: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1
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 POLYMERASES I, II, AND III SUBUNIT RPABC4
L: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4
M: AMATOXIN
N: DNA (25-MER)
P: RNA (5'-R(P*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')
T: DNA (36-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)550,92825
Polymers550,38116
Non-polymers5489
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area71450 Å2
ΔGint-433 kcal/mol
Surface area174310 Å2
MethodPISA

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Components

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DNA-DIRECTED RNA POLYMERASE II SUBUNIT ... , 6 types, 6 molecules ABCDGI

#1: Protein DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1


Mass: 217450.078 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Thymus / References: UniProt: I3LJR4, DNA-directed RNA polymerase
#2: Protein DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2


Mass: 133201.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Thymus / References: UniProt: I3LGP4, DNA-directed RNA polymerase
#3: Protein DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3


Mass: 31439.074 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Thymus / References: UniProt: I3LCH3
#4: Protein DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3


Mass: 16331.255 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Thymus / References: UniProt: A0A287ADR4
#7: Protein DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB7


Mass: 19314.283 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Thymus / References: UniProt: I3LJZ9
#9: Protein DNA-directed RNA polymerase II subunit RPB9 / 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 (pig) / Tissue: Thymus / References: UniProt: P60899

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DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT ... , 6 types, 6 molecules EFHJKL

#5: Protein DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1


Mass: 24644.318 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Thymus / References: UniProt: I3LSI7
#6: Protein DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC1


Mass: 14477.001 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Thymus / References: UniProt: F1SKN8
#8: Protein DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC3


Mass: 17162.273 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Thymus / References: UniProt: I3LCB2
#10: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC5 / RNA polymerases I / II / and III subunit ABC5 / DNA-directed RNA polymerase III subunit L


Mass: 7655.123 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus Scrofa (pig) / Tissue: Thymus
#11: Protein DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4


Mass: 13310.284 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Thymus / References: UniProt: F1RKE4
#12: Protein DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC4


Mass: 7018.244 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Tissue: Thymus / References: UniProt: I3LN51

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DNA chain , 2 types, 2 molecules NT

#14: DNA chain DNA (25-MER)


Mass: 13303.563 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#16: DNA chain DNA (36-MER)


Mass: 13178.483 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein/peptide / RNA chain , 2 types, 2 molecules MP

#13: Protein/peptide AMATOXIN


Type: Cyclic peptide / Class: Toxin / Mass: 939.004 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: ALPHA-AMANITIN, IS AN 8 AMINO-ACID PEPTIDE. THE OUTER LOOP IS FORMED BY A PEPTIDE BOND BETWEEN THE C- AND THE N-TERMINI. THE SIDE-CHAINS OF RESIDUES 2 AND 8 ARE LINKED TO FORM THE INNER LOOP.
Source: (natural) Amanita phalloides (death cap) / References: UniProt: P85421, Alpha-Amanitin
#15: RNA chain RNA (5'-R(P*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')


Mass: 6414.902 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 9 molecules

#17: Chemical
ChemComp-ZN / ZINC ION


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


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

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Details

Compound detailsALPHA-AMANITIN, AN AMATOXIN, IS A DI-CYCLIC PEPTIDE. HERE, ALPHA-AMANITIN IS REPRESENTED BY THE ...ALPHA-AMANITIN, AN AMATOXIN, IS A DI-CYCLIC PEPTIDE. HERE, ALPHA-AMANITIN IS REPRESENTED BY THE SEQUENCE (SEQRES)
Has protein modificationY

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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
1Mammalian RNA polymerase II elongation complex inhibited by Alpha-amanitinCOMPLEX#1-#160NATURAL
2RNA polymerase II elongation complexCOMPLEX#1-#121NATURAL
3Nucleic acidsCOMPLEX#14-#161RECOMBINANT
4Alpha-amanitinCOMPLEX#131NATURAL
Molecular weightValue: 0.607 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDTissue
22Sus scrofa (pig)9823Thymus
34Amanita phalloides (death cap)67723
43synthetic construct (others)32630
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.6
SpecimenConc.: 0.244 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Residual tilt: 10 mradians
Image recordingAverage exposure time: 10 sec. / Electron dose: 35 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2264
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4GctfCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 207410
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 134512 / Symmetry type: POINT
Atomic model buildingB value: 53.97 / Protocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00833491
ELECTRON MICROSCOPYf_angle_d1.06545569
ELECTRON MICROSCOPYf_dihedral_angle_d8.15420250
ELECTRON MICROSCOPYf_chiral_restr0.0635102
ELECTRON MICROSCOPYf_plane_restr0.0095669

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