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- PDB-8v38: Structure of the human systemic RNAi defective transmembrane prot... -

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Basic information

Entry
Database: PDB / ID: 8v38
TitleStructure of the human systemic RNAi defective transmembrane protein 1 (hSIDT1)
ComponentsSID1 transmembrane family member 1,RNA-directed RNA polymerase L
KeywordsMEMBRANE PROTEIN / RNA interference / transmembrane protein / cholesterol or dsRNA uptake family.
Function / homology
Function and homology information


NNS virus cap methyltransferase / RNA transmembrane transporter activity / GDP polyribonucleotidyltransferase / RNA transport / cholesterol binding / bioluminescence / generation of precursor metabolites and energy / double-stranded RNA binding / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm ...NNS virus cap methyltransferase / RNA transmembrane transporter activity / GDP polyribonucleotidyltransferase / RNA transport / cholesterol binding / bioluminescence / generation of precursor metabolites and energy / double-stranded RNA binding / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / lysosome / hydrolase activity / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / ATP binding / plasma membrane / cytoplasm
Similarity search - Function
RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / RNA-directed RNA polymerase, paramyxovirus / SID1 transmembrane family / dsRNA-gated channel SID-1 / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase ...RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / RNA-directed RNA polymerase, paramyxovirus / SID1 transmembrane family / dsRNA-gated channel SID-1 / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase / Mononegavirales mRNA-capping region V / RdRp of negative ssRNA viruses with non-segmented genomes catalytic domain profile. / Mononegavirus L protein 2'-O-ribose methyltransferase domain profile. / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
RNA-directed RNA polymerase L / SID1 transmembrane family member 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsNavratna, V. / Kumar, A. / Rana, J.K. / Mosalaganti, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: bioRxiv / Year: 2024
Title: Structure of the human systemic RNAi defective transmembrane protein 1 (hSIDT1) reveals the conformational flexibility of its lipid binding domain.
Authors: Vikas Navratna / Arvind Kumar / Jaimin K Rana / Shyamal Mosalaganti /
Abstract: In , inter-cellular transport of the small non-coding RNA causing systemic RNA interference (RNAi) is mediated by the transmembrane protein SID1, encoded by the gene in the systemic RNA interference- ...In , inter-cellular transport of the small non-coding RNA causing systemic RNA interference (RNAi) is mediated by the transmembrane protein SID1, encoded by the gene in the systemic RNA interference-defective () loci. SID1 shares structural and sequence similarity with cholesterol uptake protein 1 (CHUP1) and is classified as a member of the cholesterol uptake family (ChUP). Although systemic RNAi is not an evolutionarily conserved process, the gene products are found across the animal kingdom, suggesting the existence of other novel gene regulatory mechanisms mediated by small non-coding RNAs. Human homologs of gene products - hSIDT1 and hSIDT2 - mediate contact-dependent lipophilic small non-coding dsRNA transport. Here, we report the structure of recombinant human SIDT1. We find that the extra-cytosolic domain (ECD) of hSIDT1 adopts a double jelly roll fold, and the transmembrane domain (TMD) exists as two modules - a flexible lipid binding domain (LBD) and a rigid TMD core. Our structural analyses provide insights into the inherent conformational dynamics within the lipid binding domain in cholesterol uptake (ChUP) family members.
History
DepositionNov 27, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: SID1 transmembrane family member 1,RNA-directed RNA polymerase L
B: SID1 transmembrane family member 1,RNA-directed RNA polymerase L


Theoretical massNumber of molelcules
Total (without water)248,1962
Polymers248,1962
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: ALA / Beg label comp-ID: ALA / End auth comp-ID: VAL / End label comp-ID: VAL / Auth seq-ID: 42 - 819 / Label seq-ID: 42 - 819

Dom-IDAuth asym-IDLabel asym-ID
d_1AA
d_2BB

NCS oper: (Code: givenMatrix: (-0.999999982487, 4.20496311004E-5, -0.00018236526686), (-4.20581746387E-5, -0.999999998018, 4.68449132202E-5), (-0.000182363296687, 4.685258235E-5, 0.999999982274) ...NCS oper: (Code: given
Matrix: (-0.999999982487, 4.20496311004E-5, -0.00018236526686), (-4.20581746387E-5, -0.999999998018, 4.68449132202E-5), (-0.000182363296687, 4.685258235E-5, 0.999999982274)
Vector: 306.012540419, 306.000224991, 0.022813574541)

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Components

#1: Protein SID1 transmembrane family member 1,RNA-directed RNA polymerase L / Large structural protein / Replicase / Transcriptase


Mass: 124098.141 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SIDT1 / Plasmid: pEG BacMam C term 3C eGFP StrepII / Details (production host): Addgene # 160686 / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human)
References: UniProt: Q9NXL6, UniProt: A0A5P9VSM8, NNS virus cap methyltransferase, RNA-directed RNA polymerase, GDP polyribonucleotidyltransferase

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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

ComponentName: Human systemic RNAi defective transmembrane protein 1 (hSIDT1)
Type: COMPLEX
Details: Full length human SIDT1 expressed as a recombinant fusion protein with N-terminal GFP, in mammalian cells
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.1239 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular locationOrganelle
21Homo sapiens (human)9606Lysosomal membraneLysosomes
31Homo sapiens (human)9606
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S GnTI- / Plasmid: pEG BacMam C term StrepII eGFP 3C
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
10.5 %DigitoninC56H92O291
225 mMTrizma baseNH2C(CH2OH)31
3200 mMSodium ChlorideNaCl1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: hSIDT1 in digitonin micelle, purified by Strep-Tactin affinity chromatography
Specimen supportDetails: 15 mA current / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 17000
Image scansWidth: 5760 / Height: 4092

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3204173
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122683 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingDetails: Made using Model angelo / Source name: Other / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 116.76 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00379384
ELECTRON MICROSCOPYf_angle_d0.906312800
ELECTRON MICROSCOPYf_chiral_restr0.05181502
ELECTRON MICROSCOPYf_plane_restr0.00761632
ELECTRON MICROSCOPYf_dihedral_angle_d5.83291318
Refine LS restraints NCSType: NCS constraints / Rms dev position: 5.21703157262E-13 Å

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