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- EMDB-5556: Negative stain electron microscopy structure of Nup192 -

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Basic information

Entry
Database: EMDB / ID: EMD-5556
TitleNegative stain electron microscopy structure of Nup192
Map dataReconstruction of full-length Nup192
Sample
  • Sample: Full-length Nup192
  • Protein or peptide: Subunit of the Yeast Nuclear Pore Complex inner ring with weight 192 kDa
KeywordsNuclear Pore Complex / nuclear envelope / nuclear transport / nucleoporin
Function / homologynuclear pore inner ring / nuclear pore organization / structural constituent of nuclear pore / nucleocytoplasmic transport / nuclear pore
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsSampathkumar P / Kim SJ / Upla P / Rice W / Phillips J / Pieper U / Bonanno JB / Fernandez-Martinez J / Ketaren NE / Matsui T ...Sampathkumar P / Kim SJ / Upla P / Rice W / Phillips J / Pieper U / Bonanno JB / Fernandez-Martinez J / Ketaren NE / Matsui T / Stokes DL / Sauder JM / Burley SK / Sali A / Rout MP / Almo SC
CitationJournal: Structure / Year: 2013
Title: Structure, dynamics, evolution, and function of a major scaffold component in the nuclear pore complex.
Authors: Parthasarathy Sampathkumar / Seung Joong Kim / Paula Upla / William J Rice / Jeremy Phillips / Benjamin L Timney / Ursula Pieper / Jeffrey B Bonanno / Javier Fernandez-Martinez / Zhanna ...Authors: Parthasarathy Sampathkumar / Seung Joong Kim / Paula Upla / William J Rice / Jeremy Phillips / Benjamin L Timney / Ursula Pieper / Jeffrey B Bonanno / Javier Fernandez-Martinez / Zhanna Hakhverdyan / Natalia E Ketaren / Tsutomu Matsui / Thomas M Weiss / David L Stokes / J Michael Sauder / Stephen K Burley / Andrej Sali / Michael P Rout / Steven C Almo /
Abstract: The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure ...The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings. Nup192 is a major component of the NPC's inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2-960 [ScNup192(2-960)], which adopts an α-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2-960) could undergo long-range transition between "open" and "closed" conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2-960), and homology modeling. Evolutionary analyses using the ScNup192(2-960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.
History
DepositionDec 17, 2012-
Header (metadata) releaseDec 26, 2012-
Map releaseDec 26, 2012-
UpdateMar 26, 2014-
Current statusMar 26, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0721
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0721
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5556_1.png

Downloads & links

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Map

FileDownload / File: emd_5556.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of full-length Nup192
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.93 Å/pix.
x 80 pix.
= 234.4 Å		2.93 Å/pix.
x 80 pix.
= 234.4 Å		2.93 Å/pix.
x 80 pix.
= 234.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0721 / Movie #1: 0.0721
Minimum - Maximum-0.06163038 - 0.1534311
Average (Standard dev.)0.00213274 (±0.0170568)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 234.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.932.932.93
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z234.400234.400234.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0620.1530.002

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Supplemental data

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Sample components

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Entire : Full-length Nup192

EntireName: Full-length Nup192
Components
  • Sample: Full-length Nup192
  • Protein or peptide: Subunit of the Yeast Nuclear Pore Complex inner ring with weight 192 kDa

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Supramolecule #1000: Full-length Nup192

SupramoleculeName: Full-length Nup192 / type: sample / ID: 1000
Details: The sample was purified using affinity tags then centrifuged on a sucrose gradient. Fractions identified by SDS-PAGE and mass spectrometry.
Oligomeric state: monomer / Number unique components: 1
Molecular weightExperimental: 192 KDa / Theoretical: 192 KDa / Method: SDS-PAGE

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Macromolecule #1: Subunit of the Yeast Nuclear Pore Complex inner ring with weight ...

MacromoleculeName: Subunit of the Yeast Nuclear Pore Complex inner ring with weight 192 kDa
type: protein_or_peptide / ID: 1 / Name.synonym: ScNup192 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast / Organelle: Nucleus / Location in cell: Nuclear membrane
Molecular weightExperimental: 192 KDa / Theoretical: 192 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceGO: structural constituent of nuclear pore, nuclear pore organization, nucleocytoplasmic transport, nuclear pore, nuclear pore inner ring

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.0035 mg/mL
BufferpH: 7.4
Details: 20 mM HEPES, 300 mM NaCl, 2 mM MgCl2, 0.01% Tween 20, 0.01 mM DTT
StainingType: NEGATIVE
Details: 3 ul protein was added to grids. The sample was blotted and 3 uL 1% uranyl formate added. Uranyl formate was blotted and fresh stain added (three times). The sample was allowed to sit 30 ...Details: 3 ul protein was added to grids. The sample was blotted and 3 uL 1% uranyl formate added. Uranyl formate was blotted and fresh stain added (three times). The sample was allowed to sit 30 seconds before final blot, then air-dried.
GridDetails: 200 mesh copper grid with thin carbon support, glow discharged 20s with Gatan Solarus 20s in H2/O2.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy #1

Microscopy ID1
MicroscopeJEOL 2100F
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 250,000 times magnification
DetailsCollected on 2028x2048 CCD, 24 um/pixel
DateMay 30, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F224 (2k x 2k) / Digitization - Sampling interval: 24 µm / Number real images: 400 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Tilt angle min0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 81911 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: High tilt / Specimen holder model: JEOL / Tilt angle max: 50

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Electron microscopy #2

Microscopy ID2
MicroscopeJEOL 2100F
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 250,000 times magnification
DetailsCollected on 2028x2048 CCD, 24 um/pixel. 2 sessions are listed; data collected on multiple days between these dates
DateMar 1, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F224 (2k x 2k) / Digitization - Sampling interval: 24 µm / Number real images: 400 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Tilt angle min0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 81911 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: High tilt / Specimen holder model: JEOL / Tilt angle max: 50

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Image processing

DetailsParticles were selected manually using jweb and boxer
CTF correctionDetails: phase flip each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider, Sparx, EMAN2, EMAN
Details: Initial map was calculated by merging 3 maps obtained from random conical tilt. This was then used for 8 rounds of reference-based refinement in SPIDER at a 10-degree angular increment
Number images used: 3883
Final angle assignmentDetails: SPIDER: theta 45 degrees, phi 45 degrees
Final two d classificationNumber classes: 23

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Atomic model buiding 1

Initial modelPDB ID:

4ifq


Chain - Chain ID: A
SoftwareName: Chimera
DetailsProtocol: Rigid Body. The domains were separately fitted by manual docking followed by "Fit in Map"using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation 0.754

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