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- EMDB-34166: Human SARM1 bounded with NMN and Nanobody-C6, Conformation 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-34166
TitleHuman SARM1 bounded with NMN and Nanobody-C6, Conformation 2
Map dataLocal-refined SARM1/NMN/Nanobody-C6 complex, conformation 2
Sample
  • Complex: Human SARM1 bounded with NMN and Nanobody-C6, Conformation 2
    • Complex: SARM1
      • Protein or peptide: NAD(+) hydrolase SARM1
    • Complex: Nanobody-C6
      • Protein or peptide: Nanobody-C6
  • Ligand: BETA-NICOTINAMIDE RIBOSE MONOPHOSPHATE
KeywordsNAD(+)Hydrolase / NMN / Nanobody / HYDROLASE
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 / NADP+ nucleosidase activity / NAD catabolic process / ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase / NAD+ nucleosidase activity / protein localization to mitochondrion / NAD+ nucleotidase, cyclic ADP-ribose generating / nervous system process ...negative regulation of MyD88-independent toll-like receptor signaling pathway / MyD88-independent TLR4 cascade / Toll Like Receptor 3 (TLR3) Cascade / NADP+ nucleosidase activity / NAD catabolic process / ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase / NAD+ nucleosidase activity / protein localization to mitochondrion / NAD+ nucleotidase, cyclic ADP-ribose generating / nervous system process / Hydrolases; Glycosylases; Hydrolysing N-glycosyl compounds / regulation of dendrite morphogenesis / response to axon injury / response to glucose / regulation of neuron apoptotic process / signaling adaptor activity / 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 / mitochondrial outer membrane / microtubule / cell differentiation / axon / innate immune response / dendrite / synapse / cell surface / signal transduction / protein-containing complex / 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: 3.78 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 24, 2022-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34166.png

Downloads & links

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Map

FileDownload / File: emd_34166.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal-refined SARM1/NMN/Nanobody-C6 complex, conformation 2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 320 pix.
= 344.32 Å		1.08 Å/pix.
x 320 pix.
= 344.32 Å		1.08 Å/pix.
x 320 pix.
= 344.32 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.076 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.153
Minimum - Maximum-0.8036109 - 1.3574724
Average (Standard dev.)0.00016230138 (±0.015064746)
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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Mask #1

Fileemd_34166_msk_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34166_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34166_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Human SARM1 bounded with NMN and Nanobody-C6, Conformation 2

EntireName: Human SARM1 bounded with NMN and Nanobody-C6, Conformation 2
Components
  • Complex: Human SARM1 bounded with NMN and Nanobody-C6, Conformation 2
    • Complex: SARM1
      • Protein or peptide: NAD(+) hydrolase SARM1
    • Complex: Nanobody-C6
      • Protein or peptide: Nanobody-C6
  • Ligand: BETA-NICOTINAMIDE RIBOSE MONOPHOSPHATE

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Supramolecule #1: Human SARM1 bounded with NMN and Nanobody-C6, Conformation 2

SupramoleculeName: Human SARM1 bounded with NMN and Nanobody-C6, Conformation 2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: SARM1

SupramoleculeName: SARM1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Nanobody-C6

SupramoleculeName: Nanobody-C6 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Vicugna pacos (alpaca)

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Macromolecule #1: NAD(+) hydrolase SARM1

MacromoleculeName: NAD(+) hydrolase SARM1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / 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

UniProtKB: NAD(+) hydrolase SARM1

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Macromolecule #2: Nanobody-C6

MacromoleculeName: Nanobody-C6 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / 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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Macromolecule #3: BETA-NICOTINAMIDE RIBOSE MONOPHOSPHATE

MacromoleculeName: BETA-NICOTINAMIDE RIBOSE MONOPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: NMN
Molecular weightTheoretical: 335.227 Da
Chemical component information

ChemComp-NMN:
BETA-NICOTINAMIDE RIBOSE MONOPHOSPHATE

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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 Chloride
1.0 mMEDTAEDTA
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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.28 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3089111
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.2) / Number images used: 133097
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.1.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.2)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.1.2)

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Atomic model buiding 1

Initial model
PDB IDChain

7djt

chain_id: A, source_name: PDB, initial_model_type: experimental model

5f1k

chain_id: A, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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