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- PDB-3p8b: X-ray crystal structure of Pyrococcus furiosus transcription elon... -

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Basic information

Entry
Database: PDB / ID: 3p8b
TitleX-ray crystal structure of Pyrococcus furiosus transcription elongation factor Spt4/5
Components
  • DNA-directed RNA polymerase, subunit e''
  • Transcription antitermination protein nusG
KeywordsTRANSFERASE/TRANSCRIPTION / transcription elongation factor / RNA polymerase / TRANSFERASE-TRANSCRIPTION complex
Function / homology
Function and homology information


transcription elongation-coupled chromatin remodeling / translation elongation factor activity / regulation of DNA-templated transcription elongation / ribosome / structural constituent of ribosome / mRNA binding / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / zinc ion binding
Similarity search - Function
Rubrerythrin, domain 2 - #90 / Archaeal transcription elongation factor Spt4 / Transcription elongation factor Spt5, archaeal / Archaeal transcription elongation factor Spt4 superfamily / NusG, N-terminal domain / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / NGN domain / Transcription elongation factor SPT5 ...Rubrerythrin, domain 2 - #90 / Archaeal transcription elongation factor Spt4 / Transcription elongation factor Spt5, archaeal / Archaeal transcription elongation factor Spt4 superfamily / NusG, N-terminal domain / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / NGN domain / Transcription elongation factor SPT5 / Early transcription elongation factor of RNA pol II, NGN section / SH3 type barrels. - #30 / NusG, N-terminal / In Spt5p, this domain may confer affinity for Spt4p. It possesses a RNP-like fold. / NusG, N-terminal domain superfamily / Rubrerythrin, domain 2 / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / Single Sheet / SH3 type barrels. / Ribosomal protein L24/L26, conserved site / KOW (Kyprides, Ouzounis, Woese) motif. / Translation protein SH3-like domain superfamily / KOW motif / KOW / Ribosomal protein L2, domain 2 / Roll / Alpha-Beta Plaits / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
BETA-MERCAPTOETHANOL / Transcription elongation factor Spt5 / Transcription elongation factor Spt4
Similarity search - Component
Biological speciesPyrococcus furiosus (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsMurakami, K.S. / Klein, B.J.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2011
Title: RNA polymerase and transcription elongation factor Spt4/5 complex structure.
Authors: Brianna J Klein / Daniel Bose / Kevin J Baker / Zahirah M Yusoff / Xiaodong Zhang / Katsuhiko S Murakami /
Abstract: Spt4/5 in archaea and eukaryote and its bacterial homolog NusG is the only elongation factor conserved in all three domains of life and plays many key roles in cotranscriptional regulation and in ...Spt4/5 in archaea and eukaryote and its bacterial homolog NusG is the only elongation factor conserved in all three domains of life and plays many key roles in cotranscriptional regulation and in recruiting other factors to the elongating RNA polymerase. Here, we present the crystal structure of Spt4/5 as well as the structure of RNA polymerase-Spt4/5 complex using cryoelectron microscopy reconstruction and single particle analysis. The Spt4/5 binds in the middle of RNA polymerase claw and encloses the DNA, reminiscent of the DNA polymerase clamp and ring helicases. The transcription elongation complex model reveals that the Spt4/5 is an upstream DNA holder and contacts the nontemplate DNA in the transcription bubble. These structures reveal that the cellular RNA polymerases also use a strategy of encircling DNA to enhance its processivity as commonly observed for many nucleic acid processing enzymes including DNA polymerases and helicases.
History
DepositionOct 13, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 26, 2011Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Feb 21, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase, subunit e''
B: Transcription antitermination protein nusG
C: DNA-directed RNA polymerase, subunit e''
D: Transcription antitermination protein nusG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)52,96617
Polymers51,9064
Non-polymers1,06013
Water2,000111
1
A: DNA-directed RNA polymerase, subunit e''
B: Transcription antitermination protein nusG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,60710
Polymers25,9532
Non-polymers6548
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3610 Å2
ΔGint-13 kcal/mol
Surface area11120 Å2
MethodPISA
2
C: DNA-directed RNA polymerase, subunit e''
D: Transcription antitermination protein nusG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,3597
Polymers25,9532
Non-polymers4065
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3670 Å2
ΔGint-16 kcal/mol
Surface area11260 Å2
MethodPISA
3


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10870 Å2
ΔGint-46 kcal/mol
Surface area18790 Å2
MethodPISA
Unit cell
Length a, b, c (Å)40.532, 87.213, 133.107
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP22121

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Components

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Protein , 2 types, 4 molecules ACBD

#1: Protein DNA-directed RNA polymerase, subunit e''


Mass: 9136.414 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus (archaea) / Gene: PF0255, Spt4 / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)codonplus RIPL / References: UniProt: Q8U440
#2: Protein Transcription antitermination protein nusG


Mass: 16816.600 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus (archaea) / Gene: PF1990, Spt5 / Plasmid: pET21a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)codonplus RIPL / References: UniProt: Q8TZK1

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Non-polymers , 4 types, 124 molecules

#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#4: Chemical
ChemComp-BME / BETA-MERCAPTOETHANOL


Mass: 78.133 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C2H6OS
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 111 / Source method: isolated from a natural source / Formula: H2O

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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: 2.27 Å3/Da / Density % sol: 45.72 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: 0.1 M Bis-Tris (pH 5.5), 0.2 M NaCl, and 25% PEG3350, VAPOR DIFFUSION, SITTING DROP, temperature 295K

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Data collection

Diffraction sourceSource: SYNCHROTRON / Site: CHESS / Beamline: F1 / Wavelength: 0.9181 Å
DetectorType: ADSC QUANTUM 270 / Detector: CCD / Date: Nov 9, 2009
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9181 Å / Relative weight: 1
ReflectionResolution: 1.8→30 Å / Num. obs: 44007

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Processing

Software
NameVersionClassification
PHASERphasing
REFMAC5.5.0072refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.8→30 Å / Cor.coef. Fo:Fc: 0.945 / Cor.coef. Fo:Fc free: 0.911 / SU B: 4.424 / SU ML: 0.132 / Cross valid method: THROUGHOUT / ESU R Free: 0.154 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.29234 2208 5 %RANDOM
Rwork0.23644 ---
obs0.23928 41656 98.13 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 31.098 Å2
Baniso -1Baniso -2Baniso -3
1-0.46 Å20 Å20 Å2
2--1.09 Å20 Å2
3----1.55 Å2
Refinement stepCycle: LAST / Resolution: 1.8→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3238 0 56 111 3405
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0240.0223343
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.9911.9884499
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2465410
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.93623.433134
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.64115598
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.641524
X-RAY DIFFRACTIONr_chiral_restr0.140.2512
X-RAY DIFFRACTIONr_gen_planes_refined0.010.0212434
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.3111.52062
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it2.24423360
X-RAY DIFFRACTIONr_scbond_it3.631281
X-RAY DIFFRACTIONr_scangle_it5.9834.51139
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.8→1.847 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.407 142 -
Rwork0.396 2684 -
obs--86.26 %

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