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- EMDB-43718: NU-refined consensus map of CXCL1-KSHV ORF74-Gi-scFv16 Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-43718
TitleNU-refined consensus map of CXCL1-KSHV ORF74-Gi-scFv16 Complex
Map dataConsensus cryoEM map of KSHV ORF74-CXCL1-Gi-scFv16 complex
Sample
  • Complex: Cryo-EM Structure of KSHV ORF74-CXCL1-Gi-scFv16 Complex
    • Complex: Gi protein
    • Complex: KSHV ORF74
    • Complex: CXCL1
    • Complex: scFv16
  • Protein or peptide: Guanine nucleotide-binding protein G(i) subunit alpha-1
  • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
  • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
  • Protein or peptide: scFv Recombinant Mouse Monoclonal Antibody (scFv16) bound to ORF74-Gi
  • Protein or peptide: viral G-protein coupled receptor
  • Protein or peptide: Growth-regulated alpha protein
KeywordsKSHV vGPCR / Viral GPCR / KSHV ORF74 / CXCL1-ORF74 / ORF74 Active / CXCL1 bound ORF74 / CXCL1-KSHV ORF74-Gi-scFv16 / VIRAL PROTEIN / VIRAL PROTEIN-SIGNALING PROTEIN complex
Function / homology
Function and homology information


C-C chemokine receptor activity / C-C chemokine binding / chemokine activity / Chemokine receptors bind chemokines / Interleukin-10 signaling / adenylate cyclase inhibitor activity / positive regulation of protein localization to cell cortex / Adenylate cyclase inhibitory pathway / T cell migration / D2 dopamine receptor binding ...C-C chemokine receptor activity / C-C chemokine binding / chemokine activity / Chemokine receptors bind chemokines / Interleukin-10 signaling / adenylate cyclase inhibitor activity / positive regulation of protein localization to cell cortex / Adenylate cyclase inhibitory pathway / T cell migration / D2 dopamine receptor binding / response to prostaglandin E / adenylate cyclase regulator activity / G protein-coupled serotonin receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / cellular response to forskolin / regulation of mitotic spindle organization / cell chemotaxis / Regulation of insulin secretion / growth factor activity / enzyme activator activity / positive regulation of cholesterol biosynthetic process / calcium-mediated signaling / negative regulation of insulin secretion / G protein-coupled receptor binding / response to peptide hormone / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / centriolar satellite / Olfactory Signaling Pathway / Activation of the phototransduction cascade / specific granule lumen / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / chemotaxis / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / photoreceptor disc membrane / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / GDP binding / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADP signalling through P2Y purinoceptor 1 / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / tertiary granule lumen / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / nervous system development / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / G protein activity / positive regulation of cytosolic calcium ion concentration / GTPase binding / Ca2+ pathway / actin cytoskeleton organization / fibroblast proliferation / midbody / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / cell cortex / G alpha (i) signalling events / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell population proliferation / intracellular signal transduction / immune response / ciliary basal body / G protein-coupled receptor signaling pathway / inflammatory response / signaling receptor binding / negative regulation of cell population proliferation / lysosomal membrane / cell division / GTPase activity
Similarity search - Function
CXC chemokine / CXC chemokine, conserved site / Small cytokines (intercrine/chemokine) C-x-C subfamily signature. / CXC Chemokine domain / : / Chemokine beta/gamma/delta / Intercrine alpha family (small cytokine C-X-C) (chemokine CXC). / Chemokine interleukin-8-like domain / Chemokine interleukin-8-like superfamily / Small cytokines (intecrine/chemokine), interleukin-8 like ...CXC chemokine / CXC chemokine, conserved site / Small cytokines (intercrine/chemokine) C-x-C subfamily signature. / CXC Chemokine domain / : / Chemokine beta/gamma/delta / Intercrine alpha family (small cytokine C-X-C) (chemokine CXC). / Chemokine interleukin-8-like domain / Chemokine interleukin-8-like superfamily / Small cytokines (intecrine/chemokine), interleukin-8 like / 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 / G protein beta WD-40 repeat protein / 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. / 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
Growth-regulated alpha protein / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(i) subunit alpha-1 / viral G-protein coupled receptor
Similarity search - Component
Biological speciesHomo sapiens (human) / Human gammaherpesvirus 8 / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsSahoo B / Seo HD / Dai X / Jung J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA251275 United States
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis for ligand promiscuity and high signaling activity of Kaposi's Sarcoma-associated Herpesvirus-encoded GPCR.
Authors: Jun Bae Park / Bibekananda Sahoo / Amita Rani Sahoo / Dokyun Kim / Hogyu David Seo / James Bowman / Mi-Jeong Kwak / Sophia Suh / Matthias Buck / Xinghong Dai / Jae U Jung /
Abstract: Kaposi's Sarcoma-associated Herpesvirus encodes ORF74, a viral G protein-coupled receptor homologous to CXCR2, which plays a crucial role in Kaposi's Sarcoma development through its high basal ...Kaposi's Sarcoma-associated Herpesvirus encodes ORF74, a viral G protein-coupled receptor homologous to CXCR2, which plays a crucial role in Kaposi's Sarcoma development through its high basal signaling activity. Our cryoEM analysis of ORF74 in ligand-free, BRIL-fused ligand-free, and CXCL1/Gi-bound forms elucidates its ligand-independent signaling activity. A widely open, static extracellular cavity facilitates ligand promiscuity by enabling dynamic access and diverse binding modes. Structural alterations in CWxP, E/DRY, and NPxxY micro-switches stabilize the active conformation, leading to constitutive signaling. Metadynamics simulations reveal a dynamic ensemble between local switch structures corresponding to the inactive and active states, supporting spontaneous activation. CXCR2-ORF74 chimeras highlight intracellular loops 2 and 3 as key modulators of basal and agonist-induced activity. This study defines the structural basis of ORF74's ligand promiscuity, spontaneous activation, and high basal signaling, providing insights into its role in viral oncogenesis.
History
DepositionFeb 15, 2024-
Header (metadata) releaseSep 10, 2025-
Map releaseSep 10, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_43718.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConsensus cryoEM map of KSHV ORF74-CXCL1-Gi-scFv16 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 360 pix.
= 237.6 Å
0.66 Å/pix.
x 360 pix.
= 237.6 Å
0.66 Å/pix.
x 360 pix.
= 237.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.66 Å
Density
Contour LevelBy AUTHOR: 0.036
Minimum - Maximum-0.054838855 - 2.150468
Average (Standard dev.)0.0009840997 (±0.021472896)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 237.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A for Consensus cryoEM map of...

Fileemd_43718_half_map_1.map
AnnotationHalf map A for Consensus cryoEM map of KSHV ORF74-CXCL1-Gi-scFv16 complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map B for Consensus cryoEM map of...

Fileemd_43718_half_map_2.map
AnnotationHalf map B for Consensus cryoEM map of KSHV ORF74-CXCL1-Gi-scFv16 complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Cryo-EM Structure of KSHV ORF74-CXCL1-Gi-scFv16 Complex

EntireName: Cryo-EM Structure of KSHV ORF74-CXCL1-Gi-scFv16 Complex
Components
  • Complex: Cryo-EM Structure of KSHV ORF74-CXCL1-Gi-scFv16 Complex
    • Complex: Gi protein
    • Complex: KSHV ORF74
    • Complex: CXCL1
    • Complex: scFv16
  • Protein or peptide: Guanine nucleotide-binding protein G(i) subunit alpha-1
  • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
  • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
  • Protein or peptide: scFv Recombinant Mouse Monoclonal Antibody (scFv16) bound to ORF74-Gi
  • Protein or peptide: viral G-protein coupled receptor
  • Protein or peptide: Growth-regulated alpha protein

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Supramolecule #1: Cryo-EM Structure of KSHV ORF74-CXCL1-Gi-scFv16 Complex

SupramoleculeName: Cryo-EM Structure of KSHV ORF74-CXCL1-Gi-scFv16 Complex
type: complex / ID: 1 / Parent: 0
Molecular weightTheoretical: 49.6 KDa

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Supramolecule #2: Gi protein

SupramoleculeName: Gi protein / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: KSHV ORF74

SupramoleculeName: KSHV ORF74 / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Human gammaherpesvirus 8

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Supramolecule #4: CXCL1

SupramoleculeName: CXCL1 / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #5: scFv16

SupramoleculeName: scFv16 / type: complex / ID: 5 / Parent: 1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Guanine nucleotide-binding protein G(i) subunit alpha-1

MacromoleculeName: Guanine nucleotide-binding protein G(i) subunit alpha-1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.415031 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGCTLSAEDK AAVERSKMID RNLREDGEKA AREVKLLLLG AGESGKSTIV KQMKIIHEAG YSEEECKQYK AVVYSNTIQS IIAIIRAMG RLKIDFGDSA RADDARQLFV LAGAAEEGFM TAELAGVIKR LWKDSGVQAC FNRSREYQLN DSAAYYLNDL D RIAQPNYI ...String:
MGCTLSAEDK AAVERSKMID RNLREDGEKA AREVKLLLLG AGESGKSTIV KQMKIIHEAG YSEEECKQYK AVVYSNTIQS IIAIIRAMG RLKIDFGDSA RADDARQLFV LAGAAEEGFM TAELAGVIKR LWKDSGVQAC FNRSREYQLN DSAAYYLNDL D RIAQPNYI PTQQDVLRTR VKTTGIVETH FTFKDLHFKM FDVGGQRSER KKWIHCFEGV TAIIFCVALS DYDLVLAEDE EM NRMHESM KLFDSICNNK WFTDTSIILF LNKKDLFEEK IKKSPLTICY PEYAGSNTYE EAAAYIQCQF EDLNKRKDTK EIY THFTCA TDTKNVQFVF DAVTDVIIKN NLKDCGLF

UniProtKB: Guanine nucleotide-binding protein G(i) subunit alpha-1

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Macromolecule #2: 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: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.660293 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: HHHHHHHHLE VLFQGPPGSS GSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRL LVSASQDGKL IIWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL A GHTGYLSC ...String:
HHHHHHHHLE VLFQGPPGSS GSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRL LVSASQDGKL IIWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL A GHTGYLSC CRFLDDNQIV TSSGDTTCAL WDIETGQQTT TFTGHTGDVM SLSLAPDTRL FVSGACDASA KLWDVREGMC RQ TFTGHES DINAICFFPN GNAFATGSDD ATCRLFDLRA DQELMTYSHD NIICGITSVS FSKSGRLLLA GYDDFNCNVW DAL KADRAG VLAGHDNRVS CLGVTDDGMA VATGSWDSFL KIWN

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

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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: Homo sapiens (human)
Molecular weightTheoretical: 7.861143 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
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: scFv Recombinant Mouse Monoclonal Antibody (scFv16) bound to ORF74-Gi

MacromoleculeName: scFv Recombinant Mouse Monoclonal Antibody (scFv16) bound to ORF74-Gi
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 30.895527 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: METDTLLLWV LLLWVPGSTG DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFT ISRDDPKNTL FLQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV M TQATSSVP ...String:
METDTLLLWV LLLWVPGSTG DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFT ISRDDPKNTL FLQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV M TQATSSVP VTPGESVSIS CRSSKSLLHS NGNTYLYWFL QRPGQSPQLL IYRMSNLASG VPDRFSGSGS GTAFTLTISR LE AEDVGVY YCMQHLEYPL TFGAGTKLEL KAAALEVLFQ GPHHHHHHHH

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Macromolecule #5: viral G-protein coupled receptor

MacromoleculeName: viral G-protein coupled receptor / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human gammaherpesvirus 8
Molecular weightTheoretical: 39.601523 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAAEDFLTIF LDDDESWNET LNMSGYDYSC NFSLEVSVCE MTTVVPYTWN VGILSLIFLI NVLGNGLVTY IFCKHRSRAG AIDILLLGI CLNSLCLSIS LLAEVLMFLF PNIISTGLCR LEIFFYYLYV YLDIFSVVCV SLVRYLLVAY STRSWPKKQS L GWVLTSAA ...String:
MAAEDFLTIF LDDDESWNET LNMSGYDYSC NFSLEVSVCE MTTVVPYTWN VGILSLIFLI NVLGNGLVTY IFCKHRSRAG AIDILLLGI CLNSLCLSIS LLAEVLMFLF PNIISTGLCR LEIFFYYLYV YLDIFSVVCV SLVRYLLVAY STRSWPKKQS L GWVLTSAA WLIALVLSGD ACRHRSRVVD PVSKQAMCYE NAGNMTADWR LHVRTVSVTA GFLLPLALLI LFYALTWCVV RR TKLQARR KVRGVIVAVV VLFFVFCFPY HVLNLLDTLL RRRWIRDSCY TRGLINVGLA VTSLLQALYS AVVPLIYSCL GSL FRQRMY GLFQSLRQSF MSGADYKDDD DK

UniProtKB: viral G-protein coupled receptor

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Macromolecule #6: Growth-regulated alpha protein

MacromoleculeName: Growth-regulated alpha protein / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.131754 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
ASVATELRCQ CLQTLQGIHP KCIQSVNVKS PGPHCAQTEV IATLKNGRKA CLNPASPIVK KIIEKMLNSD KSNLEVLFQG PHHHHHHHH HH

UniProtKB: Growth-regulated alpha protein

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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

Concentration10 mg/mL
BufferpH: 7.5 / Component - Concentration: 10.0 mM / Component - Formula: C8H18N2O4S / Component - Name: HEPES
Details: 10 mM HEPES pH 7.5, 150 mM NaCl, 0.001% (w/v) LMNG, 0.0001% (w/v) CHS, 0.001% (w/v) GDN
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: NU-refinement followed by local refinement
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 190755
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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