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- EMDB-42943: Structure of the human systemic RNAi defective transmembrane prot... -

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Basic information

Entry
Database: EMDB / ID: EMD-42943
TitleStructure of the human systemic RNAi defective transmembrane protein 1 (hSIDT1)
Map dataSharpened map resulting from local refinement in cryosparc
Sample
  • Complex: Human systemic RNAi defective transmembrane protein 1 (hSIDT1)
    • Protein or peptide: SID1 transmembrane family member 1,RNA-directed RNA polymerase L
KeywordsRNA interference / transmembrane protein / cholesterol or dsRNA uptake family. / MEMBRANE PROTEIN
Function / homology
Function and homology information


RNA transmembrane transporter activity / NNS virus cap methyltransferase / RNA transport / GDP polyribonucleotidyltransferase / cholesterol binding / bioluminescence / generation of precursor metabolites and energy / virion component / double-stranded RNA binding / mRNA 5'-cap (guanine-N7-)-methyltransferase activity ...RNA transmembrane transporter activity / NNS virus cap methyltransferase / RNA transport / GDP polyribonucleotidyltransferase / cholesterol binding / bioluminescence / generation of precursor metabolites and energy / virion component / double-stranded RNA binding / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / lysosome / hydrolase activity / RNA-directed RNA polymerase / RNA-directed RNA polymerase activity / ATP binding / metal ion binding / plasma membrane
Similarity search - Function
RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / SID1 transmembrane family / dsRNA-gated channel SID-1 / RNA-directed RNA polymerase, paramyxovirus / 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 / SID1 transmembrane family / dsRNA-gated channel SID-1 / RNA-directed RNA polymerase, paramyxovirus / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsNavratna V / Kumar A / Rana JK / Mosalaganti S
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseJun 26, 2024-
Map releaseJun 26, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42943.png

Downloads & links

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Map

FileDownload / File: emd_42943.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map resulting from local refinement in cryosparc
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 360 pix.
= 306. Å		0.85 Å/pix.
x 360 pix.
= 306. Å		0.85 Å/pix.
x 360 pix.
= 306. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-1.3586525 - 1.7783748
Average (Standard dev.)0.0014587787 (±0.034027606)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 306.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: UnSharpened map resulting from local refinement in cryosparc

Fileemd_42943_additional_1.map
AnnotationUnSharpened map resulting from local refinement in cryosparc
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: Mask Used for refinement during local refinement refinement...

Fileemd_42943_additional_2.map
AnnotationMask Used for refinement during local refinement refinement of symmetry expanded particles in cryosparc
Projections & Slices
AxesZYX

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Half map: Half map resulting from Local refinement in cryosparc

Fileemd_42943_half_map_1.map
AnnotationHalf map resulting from Local refinement in cryosparc
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map resulting from Local refinement in cryosparc

Fileemd_42943_half_map_2.map
AnnotationHalf map resulting from Local refinement in cryosparc
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Human systemic RNAi defective transmembrane protein 1 (hSIDT1)

EntireName: Human systemic RNAi defective transmembrane protein 1 (hSIDT1)
Components
  • Complex: Human systemic RNAi defective transmembrane protein 1 (hSIDT1)
    • Protein or peptide: SID1 transmembrane family member 1,RNA-directed RNA polymerase L

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Supramolecule #1: Human systemic RNAi defective transmembrane protein 1 (hSIDT1)

SupramoleculeName: Human systemic RNAi defective transmembrane protein 1 (hSIDT1)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Full length human SIDT1 expressed as a recombinant fusion protein with N-terminal GFP, in mammalian cells
Source (natural)Organism: Homo sapiens (human) / Organelle: Lysosomes / Location in cell: Lysosomal membrane
Molecular weightTheoretical: 123.9 KDa

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Macromolecule #1: SID1 transmembrane family member 1,RNA-directed RNA polymerase L

MacromoleculeName: SID1 transmembrane family member 1,RNA-directed RNA polymerase L
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: NNS virus cap methyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 124.098141 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MRGCLRLALL CALPWLLLAA SPGHPAKSPR QPPAPRRDPF DAARGADFDH VYSGVVNLST ENIYSFNYTS QPDQVTAVRV YVNSSSENL NYPVLVVVRQ QKEVLSWQVP LLFQGLYQRS YNYQEVSRTL CPSEATNETG PLQQLIFVDV ASMAPLGAQY K LLVTKLKH ...String:
MRGCLRLALL CALPWLLLAA SPGHPAKSPR QPPAPRRDPF DAARGADFDH VYSGVVNLST ENIYSFNYTS QPDQVTAVRV YVNSSSENL NYPVLVVVRQ QKEVLSWQVP LLFQGLYQRS YNYQEVSRTL CPSEATNETG PLQQLIFVDV ASMAPLGAQY K LLVTKLKH FQLRTNVAFH FTASPSQPQY FLYKFPKDVD SVIIKVVSEM AYPCSVVSVQ NIMCPVYDLD HNVEFNGVYQ SM TKKAAIT LQKKDFPGEQ FFVVFVIKPE DYACGGSFFI QEKENQTWNL QRKKNLEVTI VPSIKESVYV KSSLFSVFIF LSF YLGCLL VGFVHYLRFQ RKSIDGSFGS NDGSGNMVAS HPIAASTPEG SNYGTIDESS SSPGRQMSSS DGGPPGQSDT DSSV EESDF DTMPDIESDK NIIRTKMFLY LSDLSRKDRR IVSKKYKIYF WNIITIAVFY ALPVIQLVIT YQTVVNVTGN QDICY YNFL CAHPLGVLSA FNNILSNLGH VLLGFLFLLI VLRRDILHRR ALEAKDIFAV EYGIPKHFGL FYAMGIALMM EGVLSA CYH VCPNYSNFQF DTSFMYMIAG LCMLKLYQTR HPDINASAYS AYASFAVVIM VTVLGVVFGK NDVWFWVIFS AIHVLAS LA LSTQIYYMGR FKIDLGIFRR AAMVFYTDCI QQCSRPLYMD RMVLLVVGNL VNWSFALFGL IYRPRDFASY MLGIFICN L LLYLAFYIIM KLRSSEKVLP VPLFCIVATA VMWAAALYFF FQNLSSWEGT PAESREKNRE CILLDFFDDH DIWHFLSAT ALFFSFLVLL TLDDDLDVVR RDQIPVFAAA FESRLEVLFQ GPAAAAVSKG EELFTGVVPI LVELDGDVNG HKFSVSGEGE GDATYGKLT LKFICTTGKL PVPWPTLVTT LTYGVQCFSR YPDHMKQHDF FKSAMPEGYV QERTIFFKDD GNYKTRAEVK F EGDTLVNR IELKGIDFKE DGNILGHKLE YNYNSHNVYI MADKQKNGIK VNFKIRHNIE DGSVQLADHY QQNTPIGDGP VL LPDNHYL STQSKLSKDP NEKRDHMVLL EFVTAAGITL GMDELYKSGG SGWSHPQFEK

UniProtKB: SID1 transmembrane family member 1, RNA-directed RNA polymerase L

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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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
0.5 %C56H92O29Digitonin
25.0 mMNH2C(CH2OH)3Trizma base
200.0 mMNaClSodium Chloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.026000000000000002 kPa / Details: 15 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV
DetailshSIDT1 in digitonin micelle, purified by Strep-Tactin affinity chromatography

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 17000 / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3204173
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 122683
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: in silico model / Details: Made using Model angelo
RefinementProtocol: RIGID BODY FIT
Output model

PDB-8v38:
Structure of the human systemic RNAi defective transmembrane protein 1 (hSIDT1)

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