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- PDB-6zfx: hSARM1 GraFix-ed -

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Basic information

Entry
Database: PDB / ID: 6zfx
TitlehSARM1 GraFix-ed
ComponentsNAD(+) hydrolase SARM1
KeywordsHYDROLASE / NADase / ARM domain / SAM domain / TIR domain
Function / homology
Function and homology information


extrinsic component of mitochondrial outer membrane / NAD catabolic process / negative regulation of MyD88-independent toll-like receptor signaling pathway / NAD+ nucleosidase activity / Hydrolases; Glycosylases; Hydrolysing N-glycosyl compounds / NAD+ nucleotidase, cyclic ADP-ribose generating / ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase / NAD(P)+ nucleosidase activity / signaling adaptor activity / regulation of dendrite morphogenesis ...extrinsic component of mitochondrial outer membrane / NAD catabolic process / negative regulation of MyD88-independent toll-like receptor signaling pathway / NAD+ nucleosidase activity / Hydrolases; Glycosylases; Hydrolysing N-glycosyl compounds / NAD+ nucleotidase, cyclic ADP-ribose generating / ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase / NAD(P)+ nucleosidase activity / signaling adaptor activity / regulation of dendrite morphogenesis / regulation of neuron death / response to axon injury / response to glucose / positive regulation of neuron death / microtubule / regulation of apoptotic process / synapse / axon / dendrite / innate immune response / signal transduction / mitochondrion / identical protein binding / cytosol / cytoplasm
Sterile alpha motif domain / Sterile alpha and TIR motif-containing protein 1 / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Armadillo-type fold / Toll/interleukin-1 receptor homology (TIR) domain / Sterile alpha motif/pointed domain superfamily / Armadillo-like helical
NAD(+) hydrolase SARM1
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsSporny, M. / Guez-Haddad, J. / Khazma, T. / Yaron, A. / Dessau, M. / Mim, C. / Isupov, M.N. / Zalk, R. / Hons, M. / Opatowsky, Y.
Funding support Israel, 2items
OrganizationGrant numberCountry
Israel Science Foundation1425/15 Israel
Israel Science Foundation909/19 Israel
CitationJournal: Elife / Year: 2020
Title: The structural basis for SARM1 inhibition and activation under energetic stress.
Authors: Michael Sporny / Julia Guez-Haddad / Tami Khazma / Avraham Yaron / Moshe Dessau / Yoel Shkolnisky / Carsten Mim / Michail N Isupov / Ran Zalk / Michael Hons / Yarden Opatowsky /
Abstract: SARM1 an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition, and its activation ...SARM1 an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition, and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolution. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1's own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 18, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: NAD(+) hydrolase SARM1
B: NAD(+) hydrolase SARM1
C: NAD(+) hydrolase SARM1
D: NAD(+) hydrolase SARM1
E: NAD(+) hydrolase SARM1
F: NAD(+) hydrolase SARM1
G: NAD(+) hydrolase SARM1
H: NAD(+) hydrolase SARM1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)642,46224
Polymers639,7708
Non-polymers2,69216
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14A
24E
15A
25F
16A
26G
17A
27H
18B
28C
19B
29D
110B
210E
111B
211F
112B
212G
113B
213H
114C
214D
115C
215E
116C
216F
117C
217G
118C
218H
119D
219E
120D
220F
121D
221G
122D
222H
123E
223F
124E
224G
125E
225H
126F
226G
127F
227H
128G
228H

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1111A56 - 700
2111B56 - 700
1121A56 - 700
2121C56 - 700
1131A56 - 700
2131D56 - 700
1141A56 - 700
2141E56 - 700
1151A56 - 700
2151F56 - 700
1161A56 - 700
2161G56 - 700
1171A56 - 700
2171H56 - 700
1181B56 - 700
2181C56 - 700
1191B56 - 700
2191D56 - 700
11101B56 - 700
21101E56 - 700
11111B56 - 700
21111F56 - 700
11121B56 - 700
21121G56 - 700
11131B56 - 700
21131H56 - 700
11141C56 - 700
21141D56 - 700
11151C56 - 700
21151E56 - 700
11161C56 - 700
21161F56 - 700
11171C56 - 700
21171G56 - 700
11181C56 - 700
21181H56 - 700
11191D56 - 700
21191E56 - 700
11201D56 - 700
21201F56 - 700
11211D56 - 700
21211G56 - 700
11221D56 - 700
21221H56 - 700
11231E56 - 700
21231F56 - 700
11241E56 - 700
21241G56 - 700
11251E56 - 700
21251H56 - 700
11261F56 - 700
21261G56 - 700
11271F56 - 700
21271H56 - 700
11281G56 - 700
21281H56 - 700

NCS ensembles:
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28

NCS oper:
IDCodeMatrixVector
1given(1), (), ()
2given(0.707136, 0.707078, 2.6E-5), (), ()-59.95303
3given(1), (), ()
4given(3.6E-5, 1, 2.9E-5), (), ()-0.01143
5given(1), (), ()
6given(-0.707083, 0.707131, 2.5E-5), (), ()144.71187
7given(1), (), ()
8given(-1, 4.0E-5, 3.5E-5), (), ()289.43661
9given(1), (), ()
10given(-0.707123, -0.70709, 2.8E-5), (), ()349.39047
11given(1), (), ()
12given(-8.4E-5, -1, 9.6E-5), (), ()289.44516
13given(1), (), ()
14given(0.707081, -0.707132, 2.2E-5), (), ()144.7271
15given(1), (), ()
16given(0.707103, 0.70711, 1.0E-6), (), ()-59.94668
17given(1), (), ()
18given(-7.0E-6, 1, -3.0E-6), (), ()0.00128
19given(1), (), ()
20given(-0.707108, 0.707106, 4.0E-6), (), ()144.72462
21given(1), (), ()
22given(-1, -1.8E-5, 2.0E-6), (), ()289.45175
23given(1), (), ()
24given(-0.707137, -0.707076, 5.9E-5), (), ()349.38846
25given(1), (), ()
26given(5.0E-6, -1, 1.0E-6), (), ()289.44885
27given(1), (), ()
28given(0.707105, 0.707108), (), ()-59.94722
29given(1), (), ()
30given(3.0E-6, 1), (), ()-0.00048
31given(1), (), ()
32given(-0.707116, 0.707098, 3.0E-6), (), ()144.72635
33given(1), (), ()
34given(-1, 4.8E-5, 1.6E-5), (), ()289.44116
35given(1), (), ()
36given(-0.707107, -0.707107, 8.0E-6), (), ()349.39493
37given(1), (), ()
38given(0.707111, 0.707103, -7.0E-6), (), ()-59.94587
39given(1), (), ()
40given(-1.1E-5, 1, 1.0E-6), (), ()0.00107
41given(1), (), ()
42given(-0.707072, 0.707142, -3.8E-5), (), ()144.72122
43given(1), (), ()
44given(-1, 2.0E-6, 9.0E-6), (), ()289.44772
45given(1), (), ()
46given(0.707095, 0.707119, 7.0E-6), (), ()-59.94814
47given(1), (), ()
48given(4.4E-5, 1, -6.1E-5), (), ()0.00292
49given(1), (), ()
50given(-0.707109, 0.707104, 1.4E-5), (), ()144.72336
51given(1), (), ()
52given(0.70715, 0.707064, -5.7E-5), (), ()-59.93929
53given(1), (), ()
54given(1.3E-5, 1, 1.0E-6), (), ()-0.00225
55given(1), (), ()
56given(0.707073, 0.707141, 8.0E-6), (-0.707141, 0.707073, 7.4E-5), (4.7E-5, -5.8E-5, 1)-59.94686, 144.7231, -0.00035

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Components

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


Mass: 79971.258 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SARM1, KIAA0524, SAMD2, SARM / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
References: UniProt: Q6SZW1, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase, Hydrolases; Glycosylases; Hydrolysing N-glycosyl compounds
#2: Chemical
ChemComp-S1N / (~{E})-4-methylnon-4-enedial


Mass: 168.233 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C10H16O2
Has ligand of interestN

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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: hSARM1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F
Buffer solutionpH: 8.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
1150GATAN K2 SUMMIT (4k x 4k)
2150GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0232 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 145000 / Symmetry type: POINT
RefinementResolution: 2.88→289.45 Å / Cor.coef. Fo:Fc: 0.927 / SU B: 7.025 / SU ML: 0.112 / ESU R: 0.14
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.34075 --
Obs0.34075 2126767 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 370.644 Å2
Baniso -1Baniso -2Baniso -3
1-0.07 Å20 Å20 Å2
2--0.07 Å20 Å2
3----0.14 Å2
Refinement stepCycle: 1 / Total: 39856
Refine LS restraints
Refinement-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.01240520
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.7021.63254728
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.21955064
ELECTRON MICROSCOPYr_dihedral_angle_2_deg33.33821.252240
ELECTRON MICROSCOPYr_dihedral_angle_3_deg19.723157224
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.11615384
ELECTRON MICROSCOPYr_chiral_restr0.1210.25112
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.0230744
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it14.87236.26820304
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it26.31254.36625352
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it18.73537.420216
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined72.926173914
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Number: 4959 / Refinement-ID: ELECTRON MICROSCOPY / Type: Local ncs / Weight position: 0.05015

Ens-IDDom-IDAuth asym-IDRms dev position (Å)
11A0.0001
12B0.0001
23A8.0E-5
24C8.0E-5
35A0.00011
36D0.00011
47A3.0E-5
48E3.0E-5
59A0.00011
510F0.00011
611A8.0E-5
612G8.0E-5
713A0.0001
714H0.0001
815B0.0001
816C0.0001
917B8.0E-5
918D8.0E-5
1019B0.0001
1020E0.0001
1121B3.0E-5
1122F3.0E-5
1223B0.0001
1224G0.0001
1325B8.0E-5
1326H8.0E-5
1427C0.00011
1428D0.00011
1529C8.0E-5
1530E8.0E-5
1631C0.00011
1632F0.00011
1733C1.0E-5
1734G1.0E-5
1835C0.00011
1836H0.00011
1937D0.0001
1938E0.0001
2039D8.0E-5
2040F8.0E-5
2141D0.00011
2142G0.00011
2243D2.0E-5
2244H2.0E-5
2345E0.0001
2346F0.0001
2447E8.0E-5
2448G8.0E-5
2549E0.0001
2550H0.0001
2651F0.00011
2652G0.00011
2753F8.0E-5
2754H8.0E-5
2855G0.00011
2856H0.00011
LS refinement shellResolution: 2.88→2.955 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.489 157934 -
Obs--100 %

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