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- EMDB-20629: Best fitting antiparallel model for Volume 2 of truncated dimeric... -

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Entry
Database: EMDB / ID: EMD-20629
TitleBest fitting antiparallel model for Volume 2 of truncated dimeric Cytohesin-3 (Grp1; amino acids 14-399)
Map dataVolume 2 for truncated dimeric Cytohesin-3 (Grp1; amino acids 14-399)
Sample
  • Complex: Truncated homodimer of Cytohesin-3 (Grp1, amino acids 14-399) with Inositol 1,3,4,5-tetrakis phosphate (IP4)
    • Protein or peptide: Cytohesin-3
  • Ligand: INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE
KeywordsArf GEF / Phosphoinositide binding / Sec7 Domain / PH Domain / ENDOCYTOSIS
Function / homology
Function and homology information


Golgi vesicle transport / Intra-Golgi traffic / establishment of epithelial cell polarity / regulation of ARF protein signal transduction / phosphatidylinositol-3,4,5-trisphosphate binding / bicellular tight junction / ruffle / positive regulation of cell adhesion / guanyl-nucleotide exchange factor activity / adherens junction ...Golgi vesicle transport / Intra-Golgi traffic / establishment of epithelial cell polarity / regulation of ARF protein signal transduction / phosphatidylinositol-3,4,5-trisphosphate binding / bicellular tight junction / ruffle / positive regulation of cell adhesion / guanyl-nucleotide exchange factor activity / adherens junction / Golgi membrane / nucleoplasm / plasma membrane / cytosol
Similarity search - Function
Sec7 domain / Sec7, C-terminal domain superfamily / Sec7 domain superfamily / Sec7 domain / SEC7 domain profile. / Sec7 domain / PH domain / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / PH-like domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 53.0 Å
AuthorsDas S / Lambright DG
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM056324 United States
CitationJournal: Structure / Year: 2019
Title: Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes.
Authors: Sanchaita Das / Andrew W Malaby / Agata Nawrotek / Wenhua Zhang / Mahel Zeghouf / Sarah Maslen / Mark Skehel / Srinivas Chakravarthy / Thomas C Irving / Osman Bilsel / Jacqueline Cherfils / ...Authors: Sanchaita Das / Andrew W Malaby / Agata Nawrotek / Wenhua Zhang / Mahel Zeghouf / Sarah Maslen / Mark Skehel / Srinivas Chakravarthy / Thomas C Irving / Osman Bilsel / Jacqueline Cherfils / David G Lambright /
Abstract: Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through ...Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed.
History
DepositionAug 21, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseSep 25, 2019-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6u3g
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20629.png

Downloads & links

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Map

FileDownload / File: emd_20629.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVolume 2 for truncated dimeric Cytohesin-3 (Grp1; amino acids 14-399)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.5 Å/pix.
x 80 pix.
= 520. Å		6.5 Å/pix.
x 80 pix.
= 520. Å		6.5 Å/pix.
x 80 pix.
= 520. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 6.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-9.538629999999999 - 33.090274999999998
Average (Standard dev.)0.026598023 (±0.96227825)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40-40
Dimensions808080
Spacing808080
CellA=B=C: 520.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.56.56.5
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z520.000520.000520.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-40-40-40
NC/NR/NS808080
D min/max/mean-9.53933.0900.027

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Supplemental data

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Sample components

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Entire : Truncated homodimer of Cytohesin-3 (Grp1, amino acids 14-399) wit...

EntireName: Truncated homodimer of Cytohesin-3 (Grp1, amino acids 14-399) with Inositol 1,3,4,5-tetrakis phosphate (IP4)
Components
  • Complex: Truncated homodimer of Cytohesin-3 (Grp1, amino acids 14-399) with Inositol 1,3,4,5-tetrakis phosphate (IP4)
    • Protein or peptide: Cytohesin-3
  • Ligand: INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE

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Supramolecule #1: Truncated homodimer of Cytohesin-3 (Grp1, amino acids 14-399) wit...

SupramoleculeName: Truncated homodimer of Cytohesin-3 (Grp1, amino acids 14-399) with Inositol 1,3,4,5-tetrakis phosphate (IP4)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Cytohesin-3

MacromoleculeName: Cytohesin-3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.501766 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGHHHHHHGS PEDLSLEERE ELLDIRRRKK ELIDDIERLK YEIAEVMTEI DNLTSVEESK YTQRNAQIAM GRKKFNMDPK KGIQFLIEN DLLQSSPEDV AQFLYKGEGL NKTVIGDYLG ERDDFNIKVL QAFVELHEFA DLNLVQALRQ FLWSFRLPGE A QKIDRMME ...String:
MGHHHHHHGS PEDLSLEERE ELLDIRRRKK ELIDDIERLK YEIAEVMTEI DNLTSVEESK YTQRNAQIAM GRKKFNMDPK KGIQFLIEN DLLQSSPEDV AQFLYKGEGL NKTVIGDYLG ERDDFNIKVL QAFVELHEFA DLNLVQALRQ FLWSFRLPGE A QKIDRMME AFASRYCLCN PGVFQSTDTC YVLSFAIIML NTSLHNHNVR DKPTAERFIT MNRGINEGGD LPEELLRNLY ES IKNEPFK IPEDDGNDLT YTFFNPDREG WLLKLGGRVK TWKRRWFILT DNCLYYFEYT TDKEPRGIIP LENLSIREVE DPR KPNCFE LYNPSHKGQV IKACKTEADG RVVEGNHVVY RISAPSPEEK EEWMKSIKAS ISRDPFYDML ATRKRRIANK K

UniProtKB: Cytohesin-3

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Macromolecule #2: INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE

MacromoleculeName: INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: 4IP
Molecular weightTheoretical: 500.075 Da
Chemical component information

ChemComp-4IP:
INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE

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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: 8
Component:
ConcentrationNameFormula
10.0 mMTris
150.0 mMsodium chlorideNaCl

Details: Peak fractions after gel filtration were immediately diluted, applied to freshly glow discharged grids, and stained with uranyl formate.
StainingType: NEGATIVE / Material: Uranyl Formate / Details: Stained with 0.75% (w/v) uranyl formate
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
DetailsThe sample is a uniform homodimer with significant conformational flexibility.

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Electron microscopy

MicroscopeFEI/PHILIPS CM120T
Detailsspecimen holder: FISCHIONE INSTRUMENTS DUAL AXIS TOMOGRAPHY HOLDER
Image recordingFilm or detector model: TVIPS TEMCAM-F224 (2k x 2k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number real images: 500 / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -3.2 µm / Nominal defocus min: -1.2 µm / Nominal magnification: 28000

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Image processing

DetailsImages were processed with EMAN2 after X-ray removal with IMOD
Particle selectionNumber selected: 6504 / Details: Particles were manually picked.
Startup modelType of model: NONE
Final reconstructionNumber classes used: 15 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 53.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 (ver. 2.12) / Number images used: 1863
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 15 / Avg.num./class: 124 / Software - Name: EMAN2 (ver. 2.12)

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Atomic model buiding 1

Initial modelPDB ID:

2r09


Chain - Chain ID: B / Chain - Residue range: 63-399 / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsThe model with the best correlation coefficient was selected by ADP_EM from a pool of 10000 models generated by RRT_SAMPLE using rigid bodies derived from 2R09 (autoinhibited core, amino acids 63-399) and a canonical antiparallel coiled coil (amino acids 18-53) built with CCBuilder. Flexible regions with reasonable geometry were modeled with MODELLER.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-6u3g:
Best fitting antiparallel model for Volume 2 of truncated dimeric Cytohesin-3 (Grp1; amino acids 14-399)

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