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- PDB-7nd2: Cryo-EM structure of the human FERRY complex -

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Basic information

Entry
Database: PDB / ID: 7nd2
TitleCryo-EM structure of the human FERRY complex
Components
  • Glutamine amidotransferase-like class 1 domain-containing protein 1
  • Protein phosphatase 1 regulatory subunit 21
  • Quinone oxidoreductase-like protein 1
KeywordsRNA BINDING PROTEIN / Human FERRY complex / Five-subunit Early endosome RNA and Ribosome intermediarY complex / Intracellular RNA transport / Early Endosome-associated transport of RNA
Function / homology
Function and homology information


quinone metabolic process / glyoxalase III activity / NADPH:quinone reductase activity / methylglyoxal catabolic process to D-lactate via S-lactoyl-glutathione / Oxidoreductases / NADP binding / early endosome / extracellular exosome / membrane / cytoplasm / cytosol
Similarity search - Function
Uncharacterised domain KLRAQ/TTKRSYEDQ, N-terminal / Uncharacterised domain KLRAQ/TTKRSYEDQ, C-terminal / Protein phosphatase 1 regulatory subunit 21 / Predicted coiled-coil domain-containing protein / Protein phosphatase 1 regulatory subunit 21, six-helix bundle / Predicted coiled-coil domain-containing protein / Quinone oxidoreductase-like protein 1 / Polyketide synthase, enoylreductase domain / Enoylreductase / GroES-like superfamily ...Uncharacterised domain KLRAQ/TTKRSYEDQ, N-terminal / Uncharacterised domain KLRAQ/TTKRSYEDQ, C-terminal / Protein phosphatase 1 regulatory subunit 21 / Predicted coiled-coil domain-containing protein / Protein phosphatase 1 regulatory subunit 21, six-helix bundle / Predicted coiled-coil domain-containing protein / Quinone oxidoreductase-like protein 1 / Polyketide synthase, enoylreductase domain / Enoylreductase / GroES-like superfamily / Class I glutamine amidotransferase-like / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Quinone oxidoreductase-like protein 1 / Protein phosphatase 1 regulatory subunit 21 / Glutamine amidotransferase-like class 1 domain-containing protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsQuentin, D. / Klink, B.U. / Raunser, S.
Funding support1items
OrganizationGrant numberCountry
European Research Council (ERC)615984
CitationJournal: Mol Cell / Year: 2023
Title: Structural basis of mRNA binding by the human FERRY Rab5 effector complex.
Authors: Dennis Quentin / Jan S Schuhmacher / Björn U Klink / Jeni Lauer / Tanvir R Shaikh / Pim J Huis In 't Veld / Luisa M Welp / Henning Urlaub / Marino Zerial / Stefan Raunser /
Abstract: The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals ...The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.
History
DepositionJan 29, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 2, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 14, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Assembly

Deposited unit
A: Protein phosphatase 1 regulatory subunit 21
C: Quinone oxidoreductase-like protein 1
E: Glutamine amidotransferase-like class 1 domain-containing protein 1
F: Glutamine amidotransferase-like class 1 domain-containing protein 1
D: Quinone oxidoreductase-like protein 1
B: Protein phosphatase 1 regulatory subunit 21
H: Glutamine amidotransferase-like class 1 domain-containing protein 1
G: Glutamine amidotransferase-like class 1 domain-containing protein 1


Theoretical massNumber of molelcules
Total (without water)353,8388
Polymers353,8388
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, Hydrogen-deuterium exchange mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21980 Å2
ΔGint-135 kcal/mol
Surface area87140 Å2

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Components

#1: Protein Protein phosphatase 1 regulatory subunit 21 / Coiled-coil domain-containing protein 128 / KLRAQ motif-containing protein 1


Mass: 88782.836 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP1R21, CCDC128, KLRAQ1 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): Sf-9 / References: UniProt: Q6ZMI0
#2: Protein Quinone oxidoreductase-like protein 1 / Protein 4P11 / Quinone oxidoreductase homolog 1 / QOH-1 / Zeta-crystallin homolog


Mass: 39661.414 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal His-6 tag / Source: (gene. exp.) Homo sapiens (human) / Gene: CRYZL1, 4P11 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O95825, Oxidoreductases
#3: Protein
Glutamine amidotransferase-like class 1 domain-containing protein 1 / Parkinson disease 7 domain-containing protein 1


Mass: 24237.488 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GATD1, PDDC1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8NB37

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Human FERRY (Five-subunit Early endosome RNA and Ribosome intermediarY) complexCOMPLEXall0MULTIPLE SOURCES
2Protein phosphatase 1 regulatory subunit 21COMPLEX#11RECOMBINANT
3Quinone oxidoreductase-like protein 1COMPLEX#21RECOMBINANT
4Glutamine amidotransferase-like class 1 domain-containing protein 1COMPLEX#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli BL21(DE3) (bacteria)469008
34Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5
Buffer component
IDConc.FormulaBuffer-ID
120 mMHEPES1
2250 mMNaCl1
320 mMKCl1
420 mMMgCl21
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K / Details: 3s blotting time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated defocus min: 1600 nm / Calibrated defocus max: 2800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 75.8 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1879
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
1crYOLOparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
10PHENIXmodel refinement
11Cootmodel refinement
12SPHIREinitial Euler assignment
13SPHIREfinal Euler assignment
14SPHIREclassification
15SPHIRE3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1800000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18300 / Symmetry type: POINT
Atomic model buildingDetails: To build the model for the (CRYZL1)2(PPP1r21)2(GATD1)4 core of the FERRY complex, the obtained crystal structures of CRYZL1 and GATD1 were initially fitted into the corresponding density ...Details: To build the model for the (CRYZL1)2(PPP1r21)2(GATD1)4 core of the FERRY complex, the obtained crystal structures of CRYZL1 and GATD1 were initially fitted into the corresponding density using the rigid body fitting tool in Chimera. trRosetta, a de novo protein structure prediction algorithm that is based on direct energy minimization with restrained Rosetta, was used to obtain initial models for PPP1r21. The predicted model for the 6-helix bundle domain, containing residues 246 to 498, that matched our experimental density best was subsequently fitted similar as CRYZL1 and GATD1 using rigid body fit. Manual model building for the regions N- and C-terminal 6-helix bundle, which comprise residues 218 to 245 and 499 to 552, respectively, was further guided by secondary structure predictions of individual trRosetta runs for these regions, that include the vertical helix as well as the beginning of the two terminal coiled-coils of PPP1r21. With the resulting combined model, containing residues 2 to 349, 218 to 552 and 8 to 217 of CRYZL1, PPP1r21 and GATD1, respectively, a restrained refinement in PHENIX was performed. In the next step, the model was further refined using a combination of manual building in COOT and real-space refinement in PHENIX.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01117420
ELECTRON MICROSCOPYf_angle_d1.65723660
ELECTRON MICROSCOPYf_dihedral_angle_d21.1972354
ELECTRON MICROSCOPYf_chiral_restr0.0862740
ELECTRON MICROSCOPYf_plane_restr0.0113052

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