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- EMDB-2319: Cryo-EM of full-length p97-FAF1 protein complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2319
TitleCryo-EM of full-length p97-FAF1 protein complex
Map dataReconstruction of full-length p97-FAF1 protein complex
Sample
  • Sample: Full-length p97-FAF1
  • Protein or peptide: p97
  • Protein or peptide: Fas-associated factor 1
Function / homology
Function and homology information


: / RHOH GTPase cycle / HSF1 activation / ooplasm / Translesion Synthesis by POLH / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Protein methylation / positive regulation of extrinsic apoptotic signaling pathway via death domain receptors / Ovarian tumor domain proteases ...: / RHOH GTPase cycle / HSF1 activation / ooplasm / Translesion Synthesis by POLH / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Protein methylation / positive regulation of extrinsic apoptotic signaling pathway via death domain receptors / Ovarian tumor domain proteases / Hedgehog ligand biogenesis / ABC-family proteins mediated transport / Neddylation / flavin adenine dinucleotide catabolic process / VCP-NSFL1C complex / KEAP1-NFE2L2 pathway / endosome to lysosome transport via multivesicular body sorting pathway / endoplasmic reticulum stress-induced pre-emptive quality control / cellular response to arsenite ion / BAT3 complex binding / Derlin-1 retrotranslocation complex / protein-DNA covalent cross-linking repair / cytoplasm protein quality control / positive regulation of protein K63-linked deubiquitination / positive regulation of oxidative phosphorylation / : / aggresome assembly / deubiquitinase activator activity / regulation of protein localization to chromatin / ubiquitin-modified protein reader activity / VCP-NPL4-UFD1 AAA ATPase complex / vesicle-fusing ATPase / cellular response to misfolded protein / positive regulation of mitochondrial membrane potential / negative regulation of protein localization to chromatin / K48-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / stress granule disassembly / positive regulation of ATP biosynthetic process / regulation of synapse organization / ATPase complex / ubiquitin-specific protease binding / regulation of protein catabolic process / MHC class I protein binding / polyubiquitin modification-dependent protein binding / autophagosome maturation / endoplasmic reticulum to Golgi vesicle-mediated transport / negative regulation of hippo signaling / NF-kappaB binding / translesion synthesis / regulation of cell adhesion / interstrand cross-link repair / proteasomal protein catabolic process / ATP metabolic process / ERAD pathway / heat shock protein binding / lipid droplet / Neutrophil degranulation / proteasome complex / viral genome replication / ubiquitin binding / positive regulation of DNA replication / negative regulation of smoothened signaling pathway / macroautophagy / positive regulation of protein-containing complex assembly / positive regulation of non-canonical NF-kappaB signal transduction / ADP binding / autophagy / positive regulation of protein catabolic process / cytoplasmic stress granule / positive regulation of canonical Wnt signaling pathway / nuclear envelope / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / double-strand break repair / myelin sheath / site of double-strand break / cellular response to heat / ubiquitin-dependent protein catabolic process / protein phosphatase binding / proteasome-mediated ubiquitin-dependent protein catabolic process / protein ubiquitination / ciliary basal body / protein domain specific binding / DNA repair / apoptotic process / synapse / lipid binding / ubiquitin protein ligase binding / DNA damage response / endoplasmic reticulum membrane / protein kinase binding / perinuclear region of cytoplasm / glutamatergic synapse / endoplasmic reticulum / protein-containing complex / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / nucleus
Similarity search - Function
FAS-associated factor 1-like, UBX domain / FAF1, UBA-like domain / : / Fas-associated factor 1 / UAS / : / UAS / Domain present in ubiquitin-regulatory proteins / UBX domain / UBX domain ...FAS-associated factor 1-like, UBX domain / FAF1, UBA-like domain / : / Fas-associated factor 1 / UAS / : / UAS / Domain present in ubiquitin-regulatory proteins / UBX domain / UBX domain / UBX domain profile. / UBA-like domain / AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / : / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Thioredoxin-like superfamily / Ubiquitin-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
FAS-associated factor 1 / Transitional endoplasmic reticulum ATPase
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 17.0 Å
AuthorsEwens CA / Kloppsteck P / McKeown C / Ebong I-O / Robinson C / Zhang X / Freemont P
CitationJournal: J Biol Chem / Year: 2014
Title: The p97-FAF1 protein complex reveals a common mode of p97 adaptor binding.
Authors: Caroline A Ewens / Silvia Panico / Patrik Kloppsteck / Ciaran McKeown / Ima-Obong Ebong / Carol Robinson / Xiaodong Zhang / Paul S Freemont /
Abstract: p97, also known as valosin-containing protein, is a versatile participant in the ubiquitin-proteasome system. p97 interacts with a large network of adaptor proteins to process ubiquitylated ...p97, also known as valosin-containing protein, is a versatile participant in the ubiquitin-proteasome system. p97 interacts with a large network of adaptor proteins to process ubiquitylated substrates in different cellular pathways, including endoplasmic reticulum-associated degradation and transcription factor activation. p97 and its adaptor Fas-associated factor-1 (FAF1) both have roles in the ubiquitin-proteasome system during NF-κB activation, although the mechanisms are unknown. FAF1 itself also has emerging roles in other cell-cycle pathways and displays altered expression levels in various cancer cell lines. We have performed a detailed study the p97-FAF1 interaction. We show that FAF1 binds p97 stably and in a stoichiometry of 3 to 6. Cryo-EM analysis of p97-FAF1 yielded a 17 Å reconstruction of the complex with FAF1 above the p97 ring. Characteristics of p97-FAF1 uncovered in this study reveal common features in the interactions of p97, providing mechanistic insight into how p97 mediates diverse functionalities.
History
DepositionFeb 23, 2013-
Header (metadata) releaseMar 13, 2013-
Map releaseMar 12, 2014-
UpdateMay 7, 2014-
Current statusMay 7, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0296
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0296
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2319-for-emd...

Downloads & links

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Map

FileDownload / File: emd_2319.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of full-length p97-FAF1 protein 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: 0.0296 / Movie #1: 0.0296
Minimum - Maximum-0.18447417 - 0.68788624
Average (Standard dev.)0.00144989 (±0.02399133)
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: 90.0 °

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-36-12-40
NX/NY/NZ732581
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.1840.6880.001

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Supplemental data

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Sample components

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Entire : Full-length p97-FAF1

EntireName: Full-length p97-FAF1
Components
  • Sample: Full-length p97-FAF1
  • Protein or peptide: p97
  • Protein or peptide: Fas-associated factor 1

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Supramolecule #1000: Full-length p97-FAF1

SupramoleculeName: Full-length p97-FAF1 / type: sample / ID: 1000
Oligomeric state: One hexamer of p97 bound to one trimer of FAF1
Number unique components: 2
Molecular weightTheoretical: 780 KDa / Method: Theoretical

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Macromolecule #1: p97

MacromoleculeName: p97 / type: protein_or_peptide / ID: 1 / Name.synonym: VCP / Details: full-length, wild-type / Number of copies: 1 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House Mouse
Molecular weightExperimental: 550 KDa / Theoretical: 550 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta 2 / Recombinant plasmid: PET28b
SequenceUniProtKB: Transitional endoplasmic reticulum ATPase

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Macromolecule #2: Fas-associated factor 1

MacromoleculeName: Fas-associated factor 1 / type: protein_or_peptide / ID: 2 / Details: N-terminally His6-tagged, full-length, wild-type / Number of copies: 1 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House Mouse
Molecular weightTheoretical: 230 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta2 / Recombinant plasmid: pProEx
SequenceUniProtKB: FAS-associated factor 1

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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

Concentration0.3 mg/mL
BufferpH: 7.4 / Details: 20mM HEPES, 150mM KCl
GridDetails: holey carbon, quantifoil, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: 3sec blot, 0sec drain

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureMin: 94 K / Max: 104 K / Average: 94 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateDec 20, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS / Digitization - Sampling interval: 1.76 µm / Number real images: 281 / Average electron dose: 10 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: each micrograph
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: Imagic / Number images used: 4500
Final angle assignmentDetails: Imagic
Final two d classificationNumber classes: 457

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Atomic model buiding 1

Initial modelPDB ID:

3cf2


Chain - Chain ID: a
SoftwareName: chimera, WEDA
Detailsdomains were separately fitted and taking account of symmetry
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

3qq8


Chain - #0 - Chain ID: a / Chain - #1 - Chain ID: b
SoftwareName: chimera, WEDA
Detailsdomains were separately fitted and taking account of symmetry
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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