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- EMDB-1283: Molecular architecture and conformational flexibility of human RN... -

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Basic information

Entry
Database: EMDB / ID: EMD-1283
TitleMolecular architecture and conformational flexibility of human RNA polymerase II.
Map dataConformation 1
Sample
  • Sample: human RNA Polymerase II
  • Protein or peptide: x 12 types
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 20.0 Å
AuthorsKostek SA / Grob P / De Carlo S / Lipscomb JS / Garczarek F / Nogales E
CitationJournal: Structure / Year: 2006
Title: Molecular architecture and conformational flexibility of human RNA polymerase II.
Authors: Seth A Kostek / Patricia Grob / Sacha De Carlo / J Slaton Lipscomb / Florian Garczarek / Eva Nogales /
Abstract: Transcription by RNA polymerase II (RNAPII) is a central process in eukaryotic gene regulation. While atomic details exist for the yeast RNAPII, characterization of the human complex lags behind, ...Transcription by RNA polymerase II (RNAPII) is a central process in eukaryotic gene regulation. While atomic details exist for the yeast RNAPII, characterization of the human complex lags behind, mostly due to the inability to obtain large quantities of purified material. Although the complexes have the same protein composition and high sequence similarity, understanding of transcription and of transcription-coupled DNA repair (TCR) in humans will require the use of human proteins in structural studies. We have used cryo-electron microscopy, image reconstruction, and variance analysis to characterize the structure and dynamics of human RNAPII (hRNAPII). Our studies show that hRNAPII in solution parallels the conformational flexibility of the yeast structures crystallized in different states but also illustrate a more extensive conformational range with potential biological significance. This hRNAPII study will serve as a structural platform to build up higher-order transcription and TCR complexes and to gain information that may be unique to the human RNAPII system.
History
DepositionOct 28, 2006-
Header (metadata) releaseOct 28, 2006-
Map releaseOct 28, 2006-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.32
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.32
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1283.gif

Downloads & links

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Map

FileDownload / File: emd_1283.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConformation 1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.5 Å/pix.
x 120 pix.
= 300. Å		2.5 Å/pix.
x 120 pix.
= 300. Å		2.5 Å/pix.
x 120 pix.
= 300. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.5 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.126 / Movie #1: 0.32
Minimum - Maximum-0.158582 - 0.587113
Average (Standard dev.)0.0115372 (±0.0722153)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-60
Dimensions120120120
Spacing120120120
CellA=B=C: 300 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z300.000300.000300.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-60-60-60
NC/NR/NS120120120
D min/max/mean-0.1590.5870.012

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Supplemental data

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Sample components

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Entire : human RNA Polymerase II

EntireName: human RNA Polymerase II
Components
  • Sample: human RNA Polymerase II
  • Protein or peptide: Rpb1
  • Protein or peptide: Rpb2
  • Protein or peptide: Rpb3
  • Protein or peptide: Rpb4
  • Protein or peptide: Rpb5
  • Protein or peptide: Rpb6
  • Protein or peptide: Rpb7
  • Protein or peptide: Rpb8
  • Protein or peptide: Rpb9
  • Protein or peptide: Rpb10
  • Protein or peptide: Rpb11
  • Protein or peptide: Rpb12

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Supramolecule #1000: human RNA Polymerase II

SupramoleculeName: human RNA Polymerase II / type: sample / ID: 1000 / Oligomeric state: 12 subunit complex / Number unique components: 12
Molecular weightExperimental: 500 KDa / Theoretical: 520 KDa

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Macromolecule #1: Rpb1

MacromoleculeName: Rpb1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 220 KDa

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Macromolecule #2: Rpb2

MacromoleculeName: Rpb2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 130 KDa

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Macromolecule #3: Rpb3

MacromoleculeName: Rpb3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 30 KDa

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Macromolecule #4: Rpb4

MacromoleculeName: Rpb4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 20 KDa

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Macromolecule #5: Rpb5

MacromoleculeName: Rpb5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 20 KDa

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Macromolecule #6: Rpb6

MacromoleculeName: Rpb6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 10 KDa

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Macromolecule #7: Rpb7

MacromoleculeName: Rpb7 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 20 KDa

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Macromolecule #8: Rpb8

MacromoleculeName: Rpb8 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 20 KDa

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Macromolecule #9: Rpb9

MacromoleculeName: Rpb9 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 10 KDa

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Macromolecule #10: Rpb10

MacromoleculeName: Rpb10 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 10 KDa

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Macromolecule #11: Rpb11

MacromoleculeName: Rpb11 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 10 KDa

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Macromolecule #12: Rpb12

MacromoleculeName: Rpb12 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 10 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.9
Details: 200mM (NH4)2SO4, 25mM Hepes, 0.2M EDTA, 0.05% NP-40
StainingType: NEGATIVE
Details: cryo-negative staining in a saturated solution of ammonium molybdate neutralized to a pH of 7.2. with 10 N NaOH
GridDetails: 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber temperature: 90 K / Method: stained with ammonium molybdate before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureAverage: 90 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 6.3 µm / Number real images: 19 / Average electron dose: 17 e/Å2 / Od range: 1 / Bits/pixel: 14
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50280 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 50000
Sample stageSpecimen holder: side entry / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: each micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Number images used: 3406

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