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- EMDB-33871: Cryo-EM structure of the INSL5-bound human relaxin family peptide... -

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Entry
Database: EMDB / ID: EMD-33871
TitleCryo-EM structure of the INSL5-bound human relaxin family peptidereceptor 4(RXFP4)-Gi complex
Map data
Sample
  • Complex: Cryo-EM structure of the human relaxin family peptide receptor 4 in complex with INSL5 and G protein
    • Complex: INSL5
      • Protein or peptide: Insulin-like peptide INSL5 A chain
      • Protein or peptide: Insulin-like peptide INSL5 B chain
    • Complex: human relaxin family peptide receptor 4
      • Protein or peptide: Relaxin-3 receptor 2
    • Complex: G protein
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
      • Protein or peptide: Guanine nucleotide-binding protein G(i) subunit alpha-2
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Complex: scFv16
      • Protein or peptide: scFv16
Keywordshuman relaxin family peptide receptor 4 / G protein-coupled receptor / ligand recognition / STRUCTURAL PROTEIN
Function / homology
Function and homology information


Relaxin receptors / positive regulation of feeding behavior / negative regulation of adenylate cyclase-activating adrenergic receptor signaling pathway involved in heart process / Olfactory Signaling Pathway / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G protein-coupled adenosine receptor signaling pathway / negative regulation of calcium ion-dependent exocytosis / positive regulation of urine volume / Activation of the phototransduction cascade ...Relaxin receptors / positive regulation of feeding behavior / negative regulation of adenylate cyclase-activating adrenergic receptor signaling pathway involved in heart process / Olfactory Signaling Pathway / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G protein-coupled adenosine receptor signaling pathway / negative regulation of calcium ion-dependent exocytosis / positive regulation of urine volume / Activation of the phototransduction cascade / negative regulation of adenylate cyclase activity / positive regulation of neural precursor cell proliferation / G protein-coupled peptide receptor activity / gamma-aminobutyric acid signaling pathway / negative regulation of synaptic transmission / Activation of G protein gated Potassium channels / G-protein activation / G beta:gamma signalling through PI3Kgamma / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through PLC beta / ADP signalling through P2Y purinoceptor 1 / Thromboxane signalling through TP receptor / Presynaptic function of Kainate receptors / G beta:gamma signalling through CDC42 / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Glucagon-type ligand receptors / G alpha (12/13) signalling events / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Adrenaline,noradrenaline inhibits insulin secretion / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Thrombin signalling through proteinase activated receptors (PARs) / Ca2+ pathway / Extra-nuclear estrogen signaling / G alpha (z) signalling events / G alpha (s) signalling events / G alpha (q) signalling events / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / neuronal dense core vesicle / regulation of calcium ion transport / negative regulation of apoptotic signaling pathway / neuropeptide signaling pathway / Adenylate cyclase inhibitory pathway / positive regulation of insulin receptor signaling pathway / positive regulation of vascular associated smooth muscle cell proliferation / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / response to nutrient / positive regulation of superoxide anion generation / Regulation of insulin secretion / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / hormone activity / G protein-coupled acetylcholine receptor signaling pathway / ADP signalling through P2Y purinoceptor 12 / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (z) signalling events / ADORA2B mediated anti-inflammatory cytokines production / cellular response to catecholamine stimulus / adenylate cyclase-activating dopamine receptor signaling pathway / GPER1 signaling / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / extracellular vesicle / signaling receptor complex adaptor activity / midbody / cell body / G alpha (i) signalling events / G alpha (s) signalling events / cell population proliferation / Extra-nuclear estrogen signaling / positive regulation of ERK1 and ERK2 cascade / positive regulation of cell migration / G protein-coupled receptor signaling pathway / cell division / GTPase activity / centrosome / dendrite / positive regulation of cell population proliferation / synapse / GTP binding / signal transduction / extracellular exosome / extracellular region / nucleoplasm / membrane / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
: / Angiotensin II receptor family / : / Insulin-like / Insulin/IGF/Relaxin family / Insulin / insulin-like growth factor / relaxin family. / Insulin, conserved site / Insulin family signature. / Insulin-like superfamily / G-protein alpha subunit, group I ...: / Angiotensin II receptor family / : / Insulin-like / Insulin/IGF/Relaxin family / Insulin / insulin-like growth factor / relaxin family. / Insulin, conserved site / Insulin family signature. / Insulin-like superfamily / G-protein alpha subunit, group I / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Guanine nucleotide-binding protein G(i) subunit alpha-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Relaxin-3 receptor 2 / Insulin-like peptide INSL5
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsChen Y / Zhou QT / Wang J / Xu YW / Wang Y / Yan JH / Wang YB / Zhu Q / Zhao FH / Li CH ...Chen Y / Zhou QT / Wang J / Xu YW / Wang Y / Yan JH / Wang YB / Zhu Q / Zhao FH / Li CH / Chen CW / Cai XQ / Bathgate RAD / Shen C / Liu H / Xu HE / Yang DH / Wang MW
Funding support China, 11 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81872915 China
National Natural Science Foundation of China (NSFC)82073904 China
National Natural Science Foundation of China (NSFC)82121005 China
National Natural Science Foundation of China (NSFC)81973373 China
National Natural Science Foundation of China (NSFC)82130105 China
National Natural Science Foundation of China (NSFC)21704064 China
Other government2021ZD0203400
Other government2018YFA0507000
Other governmentZDKJ2021028
Other government21JC1401600
Other government1135837
CitationJournal: Nat Commun / Year: 2023
Title: Ligand recognition mechanism of the human relaxin family peptide receptor 4 (RXFP4).
Authors: Yan Chen / Qingtong Zhou / Jiang Wang / Youwei Xu / Yun Wang / Jiahui Yan / Yibing Wang / Qi Zhu / Fenghui Zhao / Chenghao Li / Chuan-Wei Chen / Xiaoqing Cai / Ross A D Bathgate / Chun Shen ...Authors: Yan Chen / Qingtong Zhou / Jiang Wang / Youwei Xu / Yun Wang / Jiahui Yan / Yibing Wang / Qi Zhu / Fenghui Zhao / Chenghao Li / Chuan-Wei Chen / Xiaoqing Cai / Ross A D Bathgate / Chun Shen / H Eric Xu / Dehua Yang / Hong Liu / Ming-Wei Wang /
Abstract: Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and ...Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs). Here, we report three cryo-electron microscopy structures of RXFP4-G protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053. The B chain of INSL5 adopts a single α-helix that penetrates into the orthosteric pocket, while the A chain sits above the orthosteric pocket, revealing a peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. Our findings not only provide insights into ligand recognition and subtype selectivity among class A G protein-coupled receptors, but also expand the knowledge of signaling mechanisms in the insulin superfamily.
History
DepositionJul 19, 2022-
Header (metadata) releaseMar 1, 2023-
Map releaseMar 1, 2023-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_33871.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 274.176 Å
1.07 Å/pix.
x 256 pix.
= 274.176 Å
1.07 Å/pix.
x 256 pix.
= 274.176 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.071 Å
Density
Contour LevelBy AUTHOR: 0.48
Minimum - Maximum-4.245178 - 5.9669533
Average (Standard dev.)-0.0028821009 (±0.091631636)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 274.176 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33871_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_33871_half_map_2.map
Projections & Slices
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Sample components

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Entire : Cryo-EM structure of the human relaxin family peptide receptor 4 ...

EntireName: Cryo-EM structure of the human relaxin family peptide receptor 4 in complex with INSL5 and G protein
Components
  • Complex: Cryo-EM structure of the human relaxin family peptide receptor 4 in complex with INSL5 and G protein
    • Complex: INSL5
      • Protein or peptide: Insulin-like peptide INSL5 A chain
      • Protein or peptide: Insulin-like peptide INSL5 B chain
    • Complex: human relaxin family peptide receptor 4
      • Protein or peptide: Relaxin-3 receptor 2
    • Complex: G protein
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
      • Protein or peptide: Guanine nucleotide-binding protein G(i) subunit alpha-2
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Complex: scFv16
      • Protein or peptide: scFv16

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Supramolecule #1: Cryo-EM structure of the human relaxin family peptide receptor 4 ...

SupramoleculeName: Cryo-EM structure of the human relaxin family peptide receptor 4 in complex with INSL5 and G protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: INSL5

SupramoleculeName: INSL5 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: human relaxin family peptide receptor 4

SupramoleculeName: human relaxin family peptide receptor 4 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: G protein

SupramoleculeName: G protein / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4, #7
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #5: scFv16

SupramoleculeName: scFv16 / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #6
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Insulin-like peptide INSL5 A chain

MacromoleculeName: Insulin-like peptide INSL5 A chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.221531 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
QDLQTLCCTD GCSMTDLSAL C

UniProtKB: Insulin-like peptide INSL5

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Macromolecule #2: Insulin-like peptide INSL5 B chain

MacromoleculeName: Insulin-like peptide INSL5 B chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.850365 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
KESVRLCGLE YIRTVIYICA SSRW

UniProtKB: Insulin-like peptide INSL5

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Macromolecule #3: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 7.861143 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

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Macromolecule #4: Guanine nucleotide-binding protein G(i) subunit alpha-2

MacromoleculeName: Guanine nucleotide-binding protein G(i) subunit alpha-2
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.502863 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGCTVSAEDK AAAERSKMID KNLREDGEKA AREVKLLLLG AGESGKNTIV KQMKIIHEDG YSEEECRQYR AVVYSNTIQS IMAIVKAMG NLQIDFADPS RADDARQLFA LSCTAEEQGV LPDDLSGVIR RLWADHGVQA CFGRSREYQL NDSAAYYLND L ERIAQSDY ...String:
MGCTVSAEDK AAAERSKMID KNLREDGEKA AREVKLLLLG AGESGKNTIV KQMKIIHEDG YSEEECRQYR AVVYSNTIQS IMAIVKAMG NLQIDFADPS RADDARQLFA LSCTAEEQGV LPDDLSGVIR RLWADHGVQA CFGRSREYQL NDSAAYYLND L ERIAQSDY IPTQQDVLRT RVKTTGIVET HFTFKDLHFK MFDVGAQRSE RKKWIHCFEG VTAIIFCVAL SAYDLVLAED EE MNRMHAS MKLFDSICNN KWFTDTSIIL FLNKKDLFEE KITHSPLTIC FPEYTGANKY DEAASYIQSK FEDLNKRKDT KEI YTHFTC STDTKNVQFV FDAVTDVIIK NNLKDCGLF

UniProtKB: Guanine nucleotide-binding protein G(i) subunit alpha-2

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Macromolecule #5: Relaxin-3 receptor 2

MacromoleculeName: Relaxin-3 receptor 2 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.17727 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MPTLNTSASP PTFFWANASG GSVLSADDAP MPVKFLALRL MVALAYGLVG AIGLLGNLAV LWVLSNCARR APGPPSDTFV FNLALADLG LALTLPFWAA ESALDFHWPF GGALCKMVLT ATVLNVYASI FLITALSVAR YWVVAMAAGP GTHLSLFWAR I ATLAVWAA ...String:
MPTLNTSASP PTFFWANASG GSVLSADDAP MPVKFLALRL MVALAYGLVG AIGLLGNLAV LWVLSNCARR APGPPSDTFV FNLALADLG LALTLPFWAA ESALDFHWPF GGALCKMVLT ATVLNVYASI FLITALSVAR YWVVAMAAGP GTHLSLFWAR I ATLAVWAA AALVTVPTAV FGVEGEVCGV RLCLLRFPSR YWLGAYQLQR VVLAFMVPLG VITTSYLLLL AFLQRRQRRR QD SRVVARS VRILVASFFL CWFPNHVVTL WGVLVKFDLV PWNSTFYTIQ TYVFPVTTCL AHSNSCLNPV LYCLLRREPR QAL AGTFRD LRLRLWPQGG GWVQQVALKQ VGRRWVASNP RESRPSTLLT NLDRGTPG

UniProtKB: Relaxin-3 receptor 2

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Macromolecule #6: scFv16

MacromoleculeName: scFv16 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 26.408492 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SAGGGGSGGG GSGGGGSADI VMTQATSSVP VTPGESVSIS C RSSKSLLH ...String:
MVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SAGGGGSGGG GSGGGGSADI VMTQATSSVP VTPGESVSIS C RSSKSLLH SNGNTYLYWF LQRPGQSPQL LIYRMSNLAS GVPDRFSGSG SGTAFTLTIS RLEAEDVGVY YCMQHLEYPL TF GAGTKLE L

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Macromolecule #7: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.915496 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSLLQSELD QLRQEAEQLK NQIRDARKAC ADATLSQITN NIDPVGRIQM RTRRTLRGHL AKIYAMHWGT DSRLLVSASQ DGKLIIWDS YTTNKVHAIP LRSSWVMTCA YAPSGNYVAC GGLDNICSIY NLKTREGNVR VSRELAGHTG YLSCCRFLDD N QIVTSSGD ...String:
MGSLLQSELD QLRQEAEQLK NQIRDARKAC ADATLSQITN NIDPVGRIQM RTRRTLRGHL AKIYAMHWGT DSRLLVSASQ DGKLIIWDS YTTNKVHAIP LRSSWVMTCA YAPSGNYVAC GGLDNICSIY NLKTREGNVR VSRELAGHTG YLSCCRFLDD N QIVTSSGD TTCALWDIET GQQTTTFTGH TGDVMSLSLA PDTRLFVSGA CDASAKLWDV REGMCRQTFT GHESDINAIC FF PNGNAFA TGSDDATCRL FDLRADQELM TYSHDNIICG ITSVSFSKSG RLLLAGYDDF NCNVWDALKA DRAGVLAGHD NRV SCLGVT DDGMAVATGS WDSFLKIWN

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

Details: and 6D9H
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.19 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Number images used: 52435
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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