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- EMDB-34198: Human SARM1 bounded with NMN and Nanobody-C6, double-layer structure -

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Basic information

Entry
Database: EMDB / ID: EMD-34198
TitleHuman SARM1 bounded with NMN and Nanobody-C6, double-layer structure
Map dataSARM1/NMN/Nanobody-C6 double layer octameric structure
Sample
  • Complex: Human SARM1 bounded with NMN and Nanobody-C6, double-layer structure
    • Protein or peptide: NAD(+) hydrolase SARM1
    • Protein or peptide: Nanobody C6
Function / homology
Function and homology information


negative regulation of MyD88-independent toll-like receptor signaling pathway / MyD88-independent TLR4 cascade / Toll Like Receptor 3 (TLR3) Cascade / NAD catabolic process / NAD+ nucleosidase activity / ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase / protein localization to mitochondrion / NAD+ nucleotidase, cyclic ADP-ribose generating / NADP+ nucleosidase activity / nervous system process ...negative regulation of MyD88-independent toll-like receptor signaling pathway / MyD88-independent TLR4 cascade / Toll Like Receptor 3 (TLR3) Cascade / NAD catabolic process / NAD+ nucleosidase activity / ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase / protein localization to mitochondrion / NAD+ nucleotidase, cyclic ADP-ribose generating / NADP+ nucleosidase activity / nervous system process / Hydrolases; Glycosylases; Hydrolysing N-glycosyl compounds / regulation of dendrite morphogenesis / response to axon injury / response to glucose / signaling adaptor activity / regulation of neuron apoptotic process / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / IKK complex recruitment mediated by RIP1 / nervous system development / microtubule / mitochondrial outer membrane / cell differentiation / axon / innate immune response / dendrite / synapse / signal transduction / mitochondrion / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Sterile alpha and TIR motif-containing protein 1 / TIR domain / Toll - interleukin 1 - resistance / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / SAM domain (Sterile alpha motif) / SAM domain profile. / Sterile alpha motif. / Sterile alpha motif domain ...Sterile alpha and TIR motif-containing protein 1 / TIR domain / Toll - interleukin 1 - resistance / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / SAM domain (Sterile alpha motif) / SAM domain profile. / Sterile alpha motif. / Sterile alpha motif domain / Sterile alpha motif/pointed domain superfamily / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
NAD(+) hydrolase SARM1
Similarity search - Component
Biological speciesHomo sapiens (human) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsCai Y / Zhang H
Funding support China, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, China) China
CitationJournal: Nat Commun / Year: 2022
Title: A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1.
Authors: Yun Nan Hou / Yang Cai / Wan Hua Li / Wei Ming He / Zhi Ying Zhao / Wen Jie Zhu / Qiang Wang / Xinyi Mai / Jun Liu / Hon Cheung Lee / Goran Stjepanovic / Hongmin Zhang / Yong Juan Zhao /
Abstract: Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is an autoinhibitory NAD-consuming enzyme that is activated by the accumulation of nicotinamide mononucleotide ...Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is an autoinhibitory NAD-consuming enzyme that is activated by the accumulation of nicotinamide mononucleotide (NMN) during axonal injury. Its activation mechanism is not fully understood. Here, we generate a nanobody, Nb-C6, that specifically recognizes NMN-activated SARM1. Nb-C6 stains only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN, and partially activates SARM1 in vitro and in cells. Cryo-EM of NMN/SARM1/Nb-C6 complex shows an octameric structure with ARM domains bending significantly inward and swinging out together with TIR domains. Nb-C6 binds to SAM domain of the activated SARM1 and stabilized its ARM domain. Mass spectrometry analyses indicate that the activated SARM1 in solution is highly dynamic and that the neighboring TIRs form transient dimers via the surface close to one BB loop. We show that Nb-C6 is a valuable tool for studies of SARM1 activation.
History
DepositionAug 29, 2022-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateJan 18, 2023-
Current statusJan 18, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34198.png

Downloads & links

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Map

FileDownload / File: emd_34198.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSARM1/NMN/Nanobody-C6 double layer octameric structure
Voxel sizeX=Y=Z: 1.076 Å
Density
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.44758856 - 0.941245
Average (Standard dev.)-3.606185e-13 (±0.03)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 344.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_34198_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34198_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human SARM1 bounded with NMN and Nanobody-C6, double-layer structure

EntireName: Human SARM1 bounded with NMN and Nanobody-C6, double-layer structure
Components
  • Complex: Human SARM1 bounded with NMN and Nanobody-C6, double-layer structure
    • Protein or peptide: NAD(+) hydrolase SARM1
    • Protein or peptide: Nanobody C6

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Supramolecule #1: Human SARM1 bounded with NMN and Nanobody-C6, double-layer structure

SupramoleculeName: Human SARM1 bounded with NMN and Nanobody-C6, double-layer structure
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: NAD(+) hydrolase SARM1

MacromoleculeName: NAD(+) hydrolase SARM1 / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO / EC number: ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 79.486164 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MVLTLLLSAY KLCRFFAMSG PRPGAERLAV PGPDGGGGTG PWWAAGGRGP REVSPGAGTE VQDALERALP ELQQALSALK QAGGARAVG AGLAEVFQLV EEAWLLPAVG REVAQGLCDA IRLDGGLDLL LRLLQAPELE TRVQAARLLE QILVAENRDR V ARIGLGVI ...String:
MVLTLLLSAY KLCRFFAMSG PRPGAERLAV PGPDGGGGTG PWWAAGGRGP REVSPGAGTE VQDALERALP ELQQALSALK QAGGARAVG AGLAEVFQLV EEAWLLPAVG REVAQGLCDA IRLDGGLDLL LRLLQAPELE TRVQAARLLE QILVAENRDR V ARIGLGVI LNLAKEREPV ELARSVAGIL EHMFKHSEET CQRLVAAGGL DAVLYWCRRT DPALLRHCAL ALGNCALHGG QA VQRRMVE KRAAEWLFPL AFSKEDELLR LHACLAVAVL ATNKEVEREV ERSGTLALVE PLVASLDPGR FARCLVDASD TSQ GRGPDD LQRLVPLLDS NRLEAQCIGA FYLCAEAAIK SLQGKTKVFS DIGAIQSLKR LVSYSTNGTK SALAKRALRL LGEE VPRPI LPSVPSWKEA EVQTWLQQIG FSKYCESFRE QQVDGDLLLR LTEEELQTDL GMKSGITRKR FFRELTELKT FANYS TCDR SNLADWLGSL DPRFRQYTYG LVSCGLDRSL LHRVSEQQLL EDCGIHLGVH RARILTAARE MLHSPLPCTG GKPSGD TPD VFISYRRNSG SQLASLLKVH LQLHGFSVFI DVEKLEAGKF EDKLIQSVMG ARNFVLVLSP GALDKCMQDH DCKDWVH KE IVTALSCGKN IVPIIDGFEW PEPQVLPEDM QAVLTFNGIK WSHEYQEATI EKIIRFLQGR SSRDSSAGSD TSLEGAAP M GPT

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Macromolecule #2: Nanobody C6

MacromoleculeName: Nanobody C6 / type: protein_or_peptide / ID: 2 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 13.168754 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MAVQLVESGG GLVQPGGSLR LSCAASVSIS RIYVMAWYRQ APGKQREVVA VIRYDGTTNY PDSVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCNAN VETWGQGTQV TVSSHHHHHH

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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

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMTrisTris
150.0 mMNaClSodium chlorideSodium Chloride
1.0 mMEDTAEthylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV / Details: Blot time: 4s Blot force: -2.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.28 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3089111
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cisTEM (ver. 1.0.0)
Final 3D classificationSoftware - Name: cisTEM (ver. 1.0.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM (ver. 1.0.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0) / Number images used: 208299

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Atomic model buiding 1

Initial model
PDB IDChain

7djt

chain_id: A

5f1k

chain_id: A
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8gq5:
Human SARM1 bounded with NMN and Nanobody-C6, double-layer structure

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