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- EMDB-1840: The RNA polymerase and transcription elongation factor Spt4-5 com... -

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Basic information

Entry
Database: EMDB / ID: EMD-1840
TitleThe RNA polymerase and transcription elongation factor Spt4-5 complex structure
Map dataccp4 density map for PFu RNA polymerase-Spt4-Spt5 complex
Sample
  • Sample: Pyrococcus furiosus RNA polymerase and transcription elongation factor Spt4/5 complex structure
  • Protein or peptide: RNA polymerase core
  • Protein or peptide: Spt4
  • Protein or peptide: Spt5
KeywordsRNA polymerase / transcription elongation / X-ray crystallography / cryo-EM / Spt4/5-DSIF-NusG
Biological speciesPyrococcus furiosus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.0 Å
AuthorsBose D / Klein B / Baker K / Yusoff Z / Murakami K / Zhang X
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
DepositionDec 2, 2010-
Header (metadata) releaseDec 10, 2010-
Map releaseJan 7, 2011-
UpdateSep 9, 2011-
Current statusSep 9, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0005
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0005
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1840.tif

Downloads & links

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Map

FileDownload / File: emd_1840.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationccp4 density map for PFu RNA polymerase-Spt4-Spt5 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.52 Å/pix.
x 128 pix.
= 450.56 Å		3.52 Å/pix.
x 128 pix.
= 450.56 Å		3.52 Å/pix.
x 128 pix.
= 450.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.52 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0005 / Movie #1: 0.0005
Minimum - Maximum-0.00357582 - 0.00813123
Average (Standard dev.)0.00000778792 (±0.000228225)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 450.56 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.523.523.52
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z450.560450.560450.560
α/β/γ90.00090.00090.000
start NX/NY/NZ-30-24-70
NX/NY/NZ6149141
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.0040.0080.000

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Supplemental data

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Sample components

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Entire : Pyrococcus furiosus RNA polymerase and transcription elongation f...

EntireName: Pyrococcus furiosus RNA polymerase and transcription elongation factor Spt4/5 complex structure
Components
  • Sample: Pyrococcus furiosus RNA polymerase and transcription elongation factor Spt4/5 complex structure
  • Protein or peptide: RNA polymerase core
  • Protein or peptide: Spt4
  • Protein or peptide: Spt5

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Supramolecule #1000: Pyrococcus furiosus RNA polymerase and transcription elongation f...

SupramoleculeName: Pyrococcus furiosus RNA polymerase and transcription elongation factor Spt4/5 complex structure
type: sample / ID: 1000
Oligomeric state: Multisubunit RNAP core bound to heterodimer of Spt4 and Spt 5
Number unique components: 3
Molecular weightExperimental: 400 KDa / Theoretical: 400 KDa

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Macromolecule #1: RNA polymerase core

MacromoleculeName: RNA polymerase core / type: protein_or_peptide / ID: 1 / Name.synonym: RNAP core / Oligomeric state: multisubunit / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Molecular weightExperimental: 380 KDa / Theoretical: 380 KDa
Recombinant expressionOrganism: Pyrococcus furiosus (archaea)

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Macromolecule #2: Spt4

MacromoleculeName: Spt4 / type: protein_or_peptide / ID: 2 / Name.synonym: Spt4 / Number of copies: 1 / Oligomeric state: Heterodimer with Spt5 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Molecular weightExperimental: 11 KDa / Theoretical: 11 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #3: Spt5

MacromoleculeName: Spt5 / type: protein_or_peptide / ID: 3 / Name.synonym: Spt5 / Number of copies: 1 / Oligomeric state: Heterodimer with Spt4 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Molecular weightExperimental: 17 KDa / Theoretical: 17 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.1 mg/mL
BufferpH: 8
Details: 10 mM Tris-HCl (pH 8 at 4C), 0.2M KCl, 0.1 mM EDTA, 2 mM DTT, 5 mM BME
GridDetails: Glow discharged 300 mesh Quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG/ST
DetailsLow dose conditions
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Average electron dose: 10 e/Å2 / Details: CCD images / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal magnification: 50000
Sample stageSpecimen holder: Single Tilt (Gatan) / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC V, TIGRIS, EMAN / Number images used: 70890
Final angle assignmentDetails: IMAGIC alpha, beta and gamma

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Atomic model buiding 1

Initial modelPDB ID:

3hkz

SoftwareName: chimera, CNS
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

3p8b

SoftwareName: chimera, CNS
DetailsProtocol: Rigid body. X-ray structure of P. furiosus Spt4 and Spt5-NGN and Spt5-KOW were manually fitted into these densities. For fitting the Spt4 and Spt5-NGN, a hydrophobic cavity on the Spt5-NGN surface was positioned in front of a tip of the coiled-coil of the RpoA1 subunit as suggested by the site-directed and deletion mutagenesis studies. The model was refined using model-minimize.inp in CNS that carried out the conjugate gradient minimization with no experimental energy terms.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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