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- PDB-8gnj: Human SARM1 bounded with NMN and Nanobody-C6, Conformation 2 -

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Basic information

Entry
Database: PDB / ID: 8gnj
TitleHuman SARM1 bounded with NMN and Nanobody-C6, Conformation 2
Components
  • NAD(+) hydrolase SARM1
  • Nanobody-C6
KeywordsHYDROLASE / NAD(+)Hydrolase / NMN / Nanobody
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
BETA-NICOTINAMIDE RIBOSE MONOPHOSPHATE / NAD(+) hydrolase SARM1
Similarity search - Component
Biological speciesHomo sapiens (human)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsCai, Y. / Zhang, H.
Funding support China, 1items
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 24, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: NAD(+) hydrolase SARM1
B: NAD(+) hydrolase SARM1
C: Nanobody-C6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,4764
Polymers172,1413
Non-polymers3351
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein NAD(+) hydrolase SARM1 / NADase SARM1 / hSARM1 / NADP(+) hydrolase SARM1 / Sterile alpha and Armadillo repeat protein / ...NADase SARM1 / hSARM1 / NADP(+) hydrolase SARM1 / Sterile alpha and Armadillo repeat protein / Sterile alpha and TIR motif-containing protein 1 / Sterile alpha motif domain-containing protein 2 / MyD88-5 / SAM domain-containing protein 2 / Tir-1 homolog / HsTIR


Mass: 79486.164 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SARM1, KIAA0524, SAMD2, SARM / Production host: Homo sapiens (human)
References: UniProt: Q6SZW1, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase, Hydrolases; Glycosylases; Hydrolysing N-glycosyl compounds
#2: Antibody Nanobody-C6


Mass: 13168.754 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Homo sapiens (human)
#3: Chemical ChemComp-NMN / BETA-NICOTINAMIDE RIBOSE MONOPHOSPHATE / NICOTINAMIDE MONONUCLEOTIDE / Nicotinamide mononucleotide


Mass: 335.227 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H16N2O8P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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 SARM1 bounded with NMN and Nanobody-C6, Conformation 2COMPLEX#1-#20MULTIPLE SOURCES
2SARM1COMPLEX#11RECOMBINANT
3Nanobody-C6COMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Vicugna pacos (alpaca)30538
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
22Homo sapiens (human)9606HEK 293F
33Homo sapiens (human)9606
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMTrisTris1
2150 mMSodium ChlorideNaClSodium chloride1
31 mMEDTAEthylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K / Details: Blot time: 4s Blot force: -2

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.28 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4RELION3.1.2CTF correction
7PHENIX1.16model fitting
9PHENIX1.16model refinement
10RELION3.1.2initial Euler assignment
11RELION3.1.2final Euler assignment
12RELION3.1.2classification
13RELION3.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3089111
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 133097 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
17DJT

7djt

A1
25F1K

5f1k

A1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0115930
ELECTRON MICROSCOPYf_angle_d1.3338026
ELECTRON MICROSCOPYf_dihedral_angle_d10.4353606
ELECTRON MICROSCOPYf_chiral_restr0.07916
ELECTRON MICROSCOPYf_plane_restr0.0091041

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