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- EMDB-8543: 3D negative stain EM structure of Pom152, the major component of ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8543
Title3D negative stain EM structure of Pom152, the major component of the membrane ring of the nuclear pore complex
Map data3D negative stain EM structure of Pom152
Sample
  • Cell: Nucleoporin Pom152
    • Protein or peptide: Pom152
Function / homology
Function and homology information


nuclear pore transmembrane ring / spindle pole body duplication / nuclear pore organization / structural constituent of nuclear pore / nucleocytoplasmic transport / nuclear envelope lumen / mRNA transport / nuclear pore / protein-membrane adaptor activity / nuclear periphery ...nuclear pore transmembrane ring / spindle pole body duplication / nuclear pore organization / structural constituent of nuclear pore / nucleocytoplasmic transport / nuclear envelope lumen / mRNA transport / nuclear pore / protein-membrane adaptor activity / nuclear periphery / cell periphery / protein import into nucleus / nuclear envelope / nuclear membrane / mitochondrion
Similarity search - Function
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsUpla P / Kim SJ / Sampathkumar P / Dutta K / Cahill SM / Chemmama IE / Williams R / Bonanno JB / Rice WJ / Stokes DL ...Upla P / Kim SJ / Sampathkumar P / Dutta K / Cahill SM / Chemmama IE / Williams R / Bonanno JB / Rice WJ / Stokes DL / Cowburn D / Almo SC / Sali A / Rout MP / Fernandez-Martinez J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM109824 United States
CitationJournal: Structure / Year: 2017
Title: Molecular Architecture of the Major Membrane Ring Component of the Nuclear Pore Complex.
Authors: Paula Upla / Seung Joong Kim / Parthasarathy Sampathkumar / Kaushik Dutta / Sean M Cahill / Ilan E Chemmama / Rosemary Williams / Jeffrey B Bonanno / William J Rice / David L Stokes / David ...Authors: Paula Upla / Seung Joong Kim / Parthasarathy Sampathkumar / Kaushik Dutta / Sean M Cahill / Ilan E Chemmama / Rosemary Williams / Jeffrey B Bonanno / William J Rice / David L Stokes / David Cowburn / Steven C Almo / Andrej Sali / Michael P Rout / Javier Fernandez-Martinez /
Abstract: The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or ...The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or Gp210) in vertebrates. Here, we have used a combination of negative-stain electron microscopy, nuclear magnetic resonance, and small-angle X-ray scattering methods to determine an integrative structure of the ∼120 kDa luminal domain of Pom152. Our structural analysis reveals that the luminal domain is formed by a flexible string-of-pearls arrangement of nine repetitive cadherin-like Ig-like domains, indicating an evolutionary connection between NPCs and the cell adhesion machinery. The 16 copies of Pom152 known to be present in the yeast NPC are long enough to form the observed membrane ring, suggesting how interactions between Pom152 molecules help establish and maintain the NPC architecture.
History
DepositionJan 3, 2017-
Header (metadata) releaseJan 25, 2017-
Map releaseFeb 22, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0526
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0526
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8543.png

Downloads & links

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Map

FileDownload / File: emd_8543.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D negative stain EM structure of Pom152
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.93 Å/pix.
x 180 pix.
= 527.4 Å		2.93 Å/pix.
x 180 pix.
= 527.4 Å		2.93 Å/pix.
x 180 pix.
= 527.4 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: 0.0526 / Movie #1: 0.0526
Minimum - Maximum-0.07503664 - 0.16586128
Average (Standard dev.)0.0000697932 (±0.006220026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 527.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.932.932.93
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z527.400527.400527.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0750.1660.000

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Supplemental data

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Sample components

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Entire : Nucleoporin Pom152

EntireName: Nucleoporin Pom152
Components
  • Cell: Nucleoporin Pom152
    • Protein or peptide: Pom152

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Supramolecule #1: Nucleoporin Pom152

SupramoleculeName: Nucleoporin Pom152 / type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Pom152

MacromoleculeName: Pom152 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MEHRYNVFND TPRGNHWMGS SVSGSPRPSY SSRPNVNTTR RFQYSDDEPA EKIRPLRSRS FKSTESNIS DEKSRISERD SKDRYINGDK KVDIYSLPLI STDVLEISKQ RTFAVILFLI I QCYKIYDL VILKSGLPLS GLLFKNYRFN FISKYFIIDS FFLYVLPSFN ...String:
MEHRYNVFND TPRGNHWMGS SVSGSPRPSY SSRPNVNTTR RFQYSDDEPA EKIRPLRSRS FKSTESNIS DEKSRISERD SKDRYINGDK KVDIYSLPLI STDVLEISKQ RTFAVILFLI I QCYKIYDL VILKSGLPLS GLLFKNYRFN FISKYFIIDS FFLYVLPSFN IPRLTFKPWV VY LQILAML LLNIFISSDH EFVLISLIMT TWRKLYTKEL SVTGSAINHH RIFDSSAHFK GAL TIKILP ENTAMFNPLH ESYCLPMDTN LFKINSIDVP IRINSTEEIE YIELEYRDLY TNSV ELRSL SKKDFKIIDN PKSFLKKDQS VLKSHSNDFE EGSTIRYLAV TLQDIGFYQI KKIVD SKKL NLKIHQSHLV VPYCPIASIT GTGSNDRCIG DSDNVSFEIQ GVPPMKLAYS KIVNGQ TFS YVDSSLQPEY FESPLQSSKS KQSFTQGELN DLKWGRNQPV NINLDSSITQ DGKFAYK ID KITDGLGNVV DFTSLPEELK KRYDLSYNFN VHEVPRAALE ERFDPKSPTK RSIAIVFE E IKNWISDIPY VISLSYTDAQ DKSKKIMNVT TDSLTKVLQA DLPGSYNLEY IESKFCPGE IVGKSNVLVT MPVAPTMEVK SFPILDQCVG QVGLNFELSF TGAPPYYYNT KIYKLENGER KLYDAKRYT SEGTRNRFSY SPPKEGNYEI VFDTVSNKLF TEPIKLEPVK EYTFKTSMRV K PSASLKLH HDLKLCLGDH SSVPVALKGQ GPFTLTYDII ETFSSKRKTF EIKEIKTNEY VI KTPVFTT GGDYILSLVS IKDSTGCVVG LSQPDAKIQV RRDIPSAAFN FFEPIKEAKI KHG SVTEIP LKLSGEGPFT VKFKHMDYDG NIVKEFENKF QNSYKPALKV SKEGLYQLVD IRDS SCQGN VIYRNSLYKV SFLEKPKFAI QDNHHITKVT ENLFSKEEVC QGMEGTVDLA LFGSP PFIL EYDLMAPNGH ISTKKIQVAT KYASLKLPNQ IPGEYITTIK AIFDGNYGES DIHFRE HQS ELIIKQTVHP IPDVAFADGG KTLRACAANV DQISFLEPIN LKFLQGESPF SITFSVY HE STSRTDQYTI DNIDSENFSF EKLYEGMKLG NHAITIDSVV DANGCVNSLI SGPRNQIL V SITDAPKIHI LDPSTEYCVG DYVAYQLNGV APFMIKYEFN GIPLKSKERS SQFVRLASE PGIISITSLQ DSSSQCIVDF TNPKLKSEFD DLSLNIHPIP SVTVSQGNYV TEDIREGDQA EVIFSFEGT PPFSLTYVRT EETDGKHGKR RSQVVETHKV TDIYSHEYKV ITSLQGTYEA I EITDAYCF AKNDLFFNN

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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

BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMHepesHepes
300.0 mMNaClSodium Chloride
0.01 %Tween-20Polyethylene glycol sorbitan monolaurate
2.0 mMMgCl2Magnesium chloride
0.1 mMDTTDithiothreitol
PICProtease inhibitor cocktail

Details: Solutions were made fresh from concentrated, and sterile filtered.
StainingType: NEGATIVE / Material: Uranyl formate
Details: 3 ul of protein was added to grids. The excess sample was blotted and the grid washed several times with water. The sample was stained with several drops of uranyl formate, blotted between ...Details: 3 ul of protein was added to grids. The excess sample was blotted and the grid washed several times with water. The sample was stained with several drops of uranyl formate, blotted between each stain. After the last staining (30 sec), the sample was blotted and air dried.
GridModel: Electron Microscopy Sciences / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
DetailsThe endogenous sample was purified using affinity tags, then natively eluted and isolated on a 5-20% sucrose gradient. Fractions identified by SDS-PAGE and mass spectrometry.

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Electron microscopy

MicroscopeJEOL 2100F
Image recordingFilm or detector model: TVIPS TEMCAM-F224 (2k x 2k) / Number grids imaged: 2 / Number real images: 1220 / Average exposure time: 2.0 sec. / Average electron dose: 15.0 e/Å2
Details: Images were collected at tilt angles 0 and 40 degrees
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 40.0 µm / Calibrated magnification: 81911 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: JEOL

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Image processing

DetailsThe selected images were normalized.
Particle selectionNumber selected: 16826
Details: Number of particles includes 8413 untilted and 8413 tilted particles.
CTF correctionSoftware - Name: EMAN2 (ver. Ctffind3)
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Number images: 7723 / Random conical tilt - Tilt angle: 40 degrees
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 7967
Initial angle assignmentType: OTHER / Software - Name: SPARX (ver. EMAN2 2.06)
Details: Sparx was used to generate the 2D classes of the untilted particles and to get their relative orientations to derive the Euler angles, then Spider (version 17.12) was used to do the back projection.
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 1.3)
FSC plot (resolution estimation)

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