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- PDB-6xjf: X-ray crystal structure of Pyrococcus furiosus general transcript... -

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Basic information

Entry
Database: PDB / ID: 6xjf
TitleX-ray crystal structure of Pyrococcus furiosus general transcription factor TFE-alpha (SeMet labeled protein)
ComponentsTranscription factor E
KeywordsTRANSCRIPTION / TFE / general transcription factor
Function / homology
Function and homology information


transcription initiation at RNA polymerase II promoter / regulation of DNA-templated transcription / DNA binding
Similarity search - Function
Transcription factor TFE, archaea / Transcription initiation factor IIE subunit alpha, N-terminal / Transcription factor TFE/TFIIEalpha HTH domain / TFIIEalpha/SarR/Rpc3 HTH domain / Transcription factor E / TFIIE alpha subunit / TFE/IIEalpha-type HTH domain profile. / Transcription initiation factor IIE / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Transcription factor E
Similarity search - Component
Biological speciesPyrococcus furiosus (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 3.2 Å
AuthorsMurakami, K.S. / Jun, S.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM131860 United States
CitationJournal: Nat Commun / Year: 2020
Title: Direct binding of TFEα opens DNA binding cleft of RNA polymerase.
Authors: Sung-Hoon Jun / Jaekyung Hyun / Jeong Seok Cha / Hoyoung Kim / Michael S Bartlett / Hyun-Soo Cho / Katsuhiko S Murakami /
Abstract: Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron ...Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.
History
DepositionJun 23, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 8, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 20, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transcription factor E
B: Transcription factor E
C: Transcription factor E
D: Transcription factor E
E: Transcription factor E
F: Transcription factor E
G: Transcription factor E
H: Transcription factor E


Theoretical massNumber of molelcules
Total (without water)105,8278
Polymers105,8278
Non-polymers00
Water0
1
A: Transcription factor E


Theoretical massNumber of molelcules
Total (without water)13,2281
Polymers13,2281
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Transcription factor E


Theoretical massNumber of molelcules
Total (without water)13,2281
Polymers13,2281
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Transcription factor E


Theoretical massNumber of molelcules
Total (without water)13,2281
Polymers13,2281
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Transcription factor E


Theoretical massNumber of molelcules
Total (without water)13,2281
Polymers13,2281
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
5
E: Transcription factor E


Theoretical massNumber of molelcules
Total (without water)13,2281
Polymers13,2281
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
6
F: Transcription factor E


Theoretical massNumber of molelcules
Total (without water)13,2281
Polymers13,2281
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
7
G: Transcription factor E


Theoretical massNumber of molelcules
Total (without water)13,2281
Polymers13,2281
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
8
H: Transcription factor E


Theoretical massNumber of molelcules
Total (without water)13,2281
Polymers13,2281
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)76.199, 115.610, 124.209
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain "A"
21(chain "B" and resid 8 through 109)
31(chain "C" and resid 8 through 109)
41chain "D"
51(chain "E" and resid 8 through 109)
61(chain "F" and resid 8 through 109)
71(chain "G" and resid 8 through 109)
81(chain "H" and resid 8 through 109)

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
111THRGLUA1 - 102
211THRGLUB2 - 103
311THRGLUC2 - 103
411THRGLUD1 - 102
511THRGLUE2 - 103
611THRGLUF2 - 103
711THRGLUG5 - 106
811THRGLUH2 - 103

NCS oper:
IDCodeMatrixVector
1given(-0.9718613666, 0.0116108604354, -0.235267235351), (-0.106415576772, -0.912692828352, 0.394547241906), (-0.21014568549, 0.408481320253, 0.888246475858)-58.7989596629, -131.055699477, 21.1792098589
2given(0.850999179391, 0.513157768392, 0.111666921762), (0.50297879217, -0.857559649111, 0.107720856123), (0.151038840381, -0.0355042667306, -0.98789003221)55.6987730213, -148.36353994, -221.456547639
3given(-0.884048284551, -0.461792581065, -0.0721542975618), (-0.386587829286, 0.809205137408, -0.442421626778), (0.262694653222, -0.363228106933, -0.893899805069)-80.986958085, -73.3649559964, -237.959138339
4given(0.998683752875, -0.00994991607853, 0.050316606746), (-0.0454195214507, 0.284205452305, 0.957686967621), (-0.023829158936, -0.958711771102, 0.283379447268)21.4219272686, 56.6402442828, -125.653118007
5given(-0.956691485383, -0.00133542119927, -0.291100701555), (-0.287423411445, 0.162842394057, 0.943859172362), (0.046143085605, 0.986651190314, -0.15617376317)-48.3992785874, 39.8308296478, 2.1083598992
6given(0.87331041589, 0.467017273792, 0.138649859276), (0.209587783565, -0.103257045299, -0.972322448356), (-0.439774804278, 0.87819863845, -0.188056568474)71.221705308, -188.362226676, -18.9048194253
7given(-0.850915366501, -0.525048343361, -0.0163485836349), (0.219274940987, -0.32674086737, -0.919325244865), (0.477348446429, -0.785852812379, 0.393158769414)-64.8858481988, -203.622874716, -89.5874816015

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Components

#1: Protein
Transcription factor E / TFE / TFIIE subunit alpha homolog / Transcription initiation factor TFIIE


Mass: 13228.428 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1) (archaea)
Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: tfe, PF0491 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8U3H5
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.61 Å3/Da / Density % sol: 52.88 %
Crystal growTemperature: 300 K / Method: vapor diffusion, hanging drop
Details: 10 % PEG 8000, 0.2 M MgCl2, and 0.1 M Tris-HCl (pH 7)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: CHESS / Beamline: A1 / Wavelength: 0.977 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Jun 11, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.977 Å / Relative weight: 1
ReflectionResolution: 3.1→50 Å / Num. obs: 20562 / % possible obs: 99.8 % / Redundancy: 14 % / CC1/2: 0.993 / CC star: 0.998 / Rmerge(I) obs: 0.13 / Rsym value: 0.13 / Net I/σ(I): 19.818
Reflection shellResolution: 3.1→3.21 Å / Redundancy: 11.5 % / Rmerge(I) obs: 1.488 / Num. unique obs: 38448 / CC1/2: 0.671 / CC star: 0.896 / Rsym value: 1.488 / % possible all: 99.8

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
Cootmodel building
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: SAD / Resolution: 3.2→44.44 Å / SU ML: 0.5188 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 33.5969
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3037 1334 7.23 %
Rwork0.2371 17119 -
obs0.2421 18453 98.7 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 82.42 Å2
Refinement stepCycle: LAST / Resolution: 3.2→44.44 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6810 0 0 0 6810
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01356890
X-RAY DIFFRACTIONf_angle_d1.86869259
X-RAY DIFFRACTIONf_chiral_restr0.0961041
X-RAY DIFFRACTIONf_plane_restr0.01091168
X-RAY DIFFRACTIONf_dihedral_angle_d21.05382711
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
12AX-RAY DIFFRACTIONTorsion NCS1.2427494525
13AX-RAY DIFFRACTIONTorsion NCS2.17846365799
14AX-RAY DIFFRACTIONTorsion NCS1.65019864379
15AX-RAY DIFFRACTIONTorsion NCS1.28763124914
16AX-RAY DIFFRACTIONTorsion NCS1.38756777986
17AX-RAY DIFFRACTIONTorsion NCS1.66097873112
18AX-RAY DIFFRACTIONTorsion NCS2.15719073788
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2-3.310.42321160.33261633X-RAY DIFFRACTION95.21
3.31-3.450.35691420.29591663X-RAY DIFFRACTION98.69
3.45-3.60.31841260.26621690X-RAY DIFFRACTION99.4
3.6-3.790.33751360.2741711X-RAY DIFFRACTION99.57
3.79-4.030.35261250.27121659X-RAY DIFFRACTION96.96
4.03-4.340.3581270.2491702X-RAY DIFFRACTION98.39
4.34-4.780.27771360.2061729X-RAY DIFFRACTION99.47
4.78-5.470.2721360.2281731X-RAY DIFFRACTION99.68
5.47-6.880.3141430.26231759X-RAY DIFFRACTION99.79
6.89-44.440.2521470.19011842X-RAY DIFFRACTION99.7

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