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- EMDB-23997: Structure of the mammalian Transport Protein Particle Complex (TR... -

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Basic information

Entry
Database: EMDB / ID: EMD-23997
TitleStructure of the mammalian Transport Protein Particle Complex (TRAPP) III
Map data
Sample
  • Complex: Transport Protein Particle Complex III (TRAPP III)
Function / homology
Function and homology information


constitutive secretory pathway / regulation of kinetochore assembly / vesicle coating / vesicle tethering / positive regulation of protein localization to kinetochore / TRAPPII protein complex / TRAPPIII protein complex / TRAPP complex / collagen biosynthetic process / metaphase chromosome alignment ...constitutive secretory pathway / regulation of kinetochore assembly / vesicle coating / vesicle tethering / positive regulation of protein localization to kinetochore / TRAPPII protein complex / TRAPPIII protein complex / TRAPP complex / collagen biosynthetic process / metaphase chromosome alignment / regulation of protein complex stability / COPII vesicle coating / RAB GEFs exchange GTP for GDP on RABs / Golgi organization / endoplasmic reticulum-Golgi intermediate compartment / endoplasmic reticulum to Golgi vesicle-mediated transport / kinetochore / Golgi apparatus / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
Trafficking protein particle complex subunit 13 / Protein of unknown function (DUF974) / Trafficking protein particle complex subunit 11 / Trafficking protein particle complex subunit 11, C-terminal / Foie gras liver health family 1 / Gryzun, putative Golgi trafficking / TRAPP III complex, Trs85 / ER-Golgi trafficking TRAPP I complex 85 kDa subunit / Tetratricopeptide repeat / Tetratricopeptide repeat ...Trafficking protein particle complex subunit 13 / Protein of unknown function (DUF974) / Trafficking protein particle complex subunit 11 / Trafficking protein particle complex subunit 11, C-terminal / Foie gras liver health family 1 / Gryzun, putative Golgi trafficking / TRAPP III complex, Trs85 / ER-Golgi trafficking TRAPP I complex 85 kDa subunit / Tetratricopeptide repeat / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
Trafficking protein particle complex subunit 13 / Trafficking protein particle complex subunit 11 / Trafficking protein particle complex subunit 12 / Trafficking protein particle complex subunit 8
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 14.2 Å
AuthorsHarris NJ / Dalwadi U / Yip CK / Burke JE / Jenkins ML
Funding support Canada, 4 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)2020-04241 Canada
Other private17868 Canada
Canadian Institutes of Health Research (CIHR)FDN-143228 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2018-03951 Canada
CitationJournal: J Mol Biol / Year: 2021
Title: Biochemical Insight into Novel Rab-GEF Activity of the Mammalian TRAPPIII Complex.
Authors: Noah J Harris / Meredith L Jenkins / Udit Dalwadi / Kaelin D Fleming / Sung-Eun Nam / Matthew A H Parson / Calvin K Yip / John E Burke /
Abstract: Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab ...Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.
History
DepositionMay 13, 2021-
Header (metadata) releaseJul 28, 2021-
Map releaseJul 28, 2021-
UpdateAug 4, 2021-
Current statusAug 4, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.669
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.669
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23997.png

Downloads & links

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Map

FileDownload / File: emd_23997.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.27 Å/pix.
x 296 pix.
= 671.92 Å		2.27 Å/pix.
x 296 pix.
= 671.92 Å		2.27 Å/pix.
x 296 pix.
= 671.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.27 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.669 / Movie #1: 0.669
Minimum - Maximum-2.152202 - 8.145609
Average (Standard dev.)-0.0136373965 (±0.21634513)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions296296296
Spacing296296296
CellA=B=C: 671.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.272.272.27
M x/y/z296296296
origin x/y/z0.0000.0000.000
length x/y/z671.920671.920671.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS296296296
D min/max/mean-2.1528.146-0.014

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Supplemental data

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Sample components

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Entire : Transport Protein Particle Complex III (TRAPP III)

EntireName: Transport Protein Particle Complex III (TRAPP III)
Components
  • Complex: Transport Protein Particle Complex III (TRAPP III)

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Supramolecule #1: Transport Protein Particle Complex III (TRAPP III)

SupramoleculeName: Transport Protein Particle Complex III (TRAPP III) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Mammalian TRAPP III purified from Sf9 insect cells
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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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

Concentration0.02 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
100.0 mMSodium ChlorideNaCl
0.5 mMTris(2-carboxyethyl)phosphine

Details: Freshly prepared gel filtration buffer, filtered through 0.22um filter and degassed
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Negative stained EM samples prepared by adsorbing sample on grid for 15s, followed by blotting of sample, 2 washes with water, 1 wash with stain and a final 30s soak in stain.
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
Details: Glow discharged using a Pelco EasiGlow. 15mA current.
DetailsTRAPP III Complex was purified to homogeneity using affinity chromatography followed by size exclusion chromatography. Samples were kept at 4 degrees and shipped on wet ice for single particle EM analysis.

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Electron microscopy

MicroscopeTFS TALOS L120C
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 2.27 µm / Number grids imaged: 1 / Number real images: 100 / Average exposure time: 1.0 sec. / Average electron dose: 5.0 e/Å2
Details: Images were manually collected every 200nm on a grid square after a 30s delay for stage stabilization. Focus was calibrated every 10 images using thin rings in the power spectra.
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus min: 1.2 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos L120C / Image courtesy: FEI Company

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Image processing

DetailsImages were first imported to RELION 3.1 for pre-processing and particle picking
Particle selectionNumber selected: 38213
Details: 200 particles were manually picked and classified into 4 classes to generate a template. Templates were then used for automated picking using the RELION reference-based auto picker.
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Details: CTF estimation was carried out using all default settings in CTFFIND4, CTF correction carried out during 2D and 3D classification
Startup modelType of model: NONE
Details: Ab initio reconstruction using cryoSPARC v2.14 ab initio reconstruction job set to generate 2 classes
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 14.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14) / Number images used: 32429
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14) / Details: cryoSPARC v2.14 ab initio reconstruction job
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14)
Details: cryoSPARC v2.14 homogenous refinement job, default settings
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 2.14)
Details: Ab initio reconstruction of 2 classes followed by heterogenous refinement using the models generated in cryoSPARC v2.14
FSC plot (resolution estimation)

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