- EMDB-21435: Single Particle Cryo-EM Structure of the Natively Isolated Sec61,... -
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Basic information
Entry
Database: EMDB / ID: EMD-21435
Title
Single Particle Cryo-EM Structure of the Natively Isolated Sec61, TMCO1, Nicalin, TMEM147, and CCDC47 Containing Ribosome-Translocon Complex After Focused Refinement with Local Angular Searches
Map data
Sample
Complex: Native complex of a translating ribosome and a novel translocon containing the Sec61 complex, TMEM147, NOMO, Nicalin, CCDC47, and TMCO1.
Complex: 60S Subunit
Complex: 60S acidic ribosomal protein P0
Complex: 60S acidic ribosomal protein P1
Complex: 60S acidic ribosomal protein P2
Complex: 60S ribosomal protein L10
Complex: 60S ribosomal protein L10a
Complex: 60S ribosomal protein L11
Complex: 60S ribosomal protein L12
Complex: 60S ribosomal protein L13
Complex: 60S ribosomal protein L13a
Complex: 60S ribosomal protein L14
Complex: 60S ribosomal protein L15
Complex: 60S ribosomal protein L17
Complex: 60S ribosomal protein L18
Complex: 60S ribosomal protein L18a
Complex: 60S ribosomal protein L19
Complex: 60S ribosomal protein L21
Complex: 60S ribosomal protein L22
Complex: 60S ribosomal protein L23
Complex: 60S ribosomal protein L23a
Complex: 60S ribosomal protein L24
Complex: 60S ribosomal protein L26
Complex: 60S ribosomal protein L27
Complex: 60S ribosomal protein L27a
Complex: 60S ribosomal protein L28
Complex: 60S ribosomal protein L3
Complex: 60S ribosomal protein L30
Complex: 60S ribosomal protein L31
Complex: 60S ribosomal protein L32
Complex: 60S ribosomal protein L34
Complex: 60S ribosomal protein L35
Complex: 60S ribosomal protein L35a
Complex: 60S ribosomal protein L36
Complex: 60S ribosomal protein L36a
Complex: 60S ribosomal protein L37a
Complex: 60S ribosomal protein L38
Complex: 60S ribosomal protein L39
Complex: 60S ribosomal protein L4
Complex: 60S ribosomal protein L5
Complex: 60S ribosomal protein L6
Complex: 60S ribosomal protein L7
Complex: 60S ribosomal protein L7a
Complex: 60S ribosomal protein L8
Complex: 60S ribosomal protein L9
Complex: 40S Subunit
Complex: 40S ribosomal protein S10
Complex: 40S ribosomal protein S11
Complex: 40S ribosomal protein S12
Complex: 40S ribosomal protein S13
Complex: 40S ribosomal protein S14
Complex: 40S ribosomal protein S15
Complex: 40S ribosomal protein S15a
Complex: 40S ribosomal protein S16
Complex: 40S ribosomal protein S17
Complex: 40S ribosomal protein S18
Complex: 40S ribosomal protein S19
Complex: 40S ribosomal protein S2
Complex: 40S ribosomal protein S20
Complex: 40S ribosomal protein S21
Complex: 40S ribosomal protein S23
Complex: 40S ribosomal protein S24
Complex: 40S ribosomal protein S25
Complex: 40S ribosomal protein S26
Complex: 40S ribosomal protein S27
Complex: 40S ribosomal protein S27-like
Complex: 40S ribosomal protein S28
Complex: 40S ribosomal protein S29
Complex: 40S ribosomal protein S3
Complex: 40S ribosomal protein S30
Complex: 40S ribosomal protein S3a
Complex: 40S ribosomal protein S4, X isoform
Complex: 40S ribosomal protein S5
Complex: 40S ribosomal protein S6
Complex: 40S ribosomal protein S7
Complex: 40S ribosomal protein S8
Complex: 40S ribosomal protein S9
Complex: 40S ribosomal protein SA
Complex: Translocon
Complex: Sec61 Complex
Complex: TMCO1
Complex: NOMO-Nicalin-TMEM147 Complex
Complex: CCDC47
Function / homology
Function and homology information
: / : / endoplasmic reticulum Sec complex / pronephric nephron development / endoplasmic reticulum quality control compartment / rough endoplasmic reticulum membrane / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER ...: / : / endoplasmic reticulum Sec complex / pronephric nephron development / endoplasmic reticulum quality control compartment / rough endoplasmic reticulum membrane / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein insertion into ER membrane / regulation of protein complex stability / embryonic brain development / eukaryotic 80S initiation complex / post-translational protein targeting to endoplasmic reticulum membrane / negative regulation of protein neddylation / translation at presynapse / SRP-dependent cotranslational protein targeting to membrane / axial mesoderm development / negative regulation of formation of translation preinitiation complex / 90S preribosome assembly / signal sequence binding / TORC2 complex binding / endoplasmic reticulum organization / endoplasmic reticulum calcium ion homeostasis / post-translational protein targeting to membrane, translocation / GAIT complex / SRP-dependent cotranslational protein targeting to membrane, translocation / middle ear morphogenesis / cytoplasmic side of rough endoplasmic reticulum membrane / A band / alpha-beta T cell differentiation / exit from mitosis / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / regulation of translation involved in cellular response to UV / positive regulation of DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator / epidermal growth factor binding / optic nerve development / negative regulation of ubiquitin protein ligase activity / retrograde protein transport, ER to cytosol / response to aldosterone / retinal ganglion cell axon guidance / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / homeostatic process / lung morphogenesis / ER overload response / Protein hydroxylation / macrophage chemotaxis / Peptide chain elongation / Selenocysteine synthesis / positive regulation of signal transduction by p53 class mediator / Formation of a pool of free 40S subunits / ubiquitin ligase inhibitor activity / Eukaryotic Translation Termination / blastocyst development / Response of EIF2AK4 (GCN2) to amino acid deficiency / SRP-dependent cotranslational protein targeting to membrane / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Viral mRNA Translation / protein localization to nucleus / L13a-mediated translational silencing of Ceruloplasmin expression / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / GTP hydrolysis and joining of the 60S ribosomal subunit / protein transmembrane transporter activity / regulation of signal transduction / regulation of protein-containing complex assembly / protein-RNA complex assembly / Major pathway of rRNA processing in the nucleolus and cytosol / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / protein targeting / cellular response to interleukin-4 / translation regulator activity / cytosolic ribosome / ERAD pathway / rough endoplasmic reticulum / MDM2/MDM4 family protein binding / DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest / embryo implantation / negative regulation of ubiquitin-dependent protein catabolic process / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ossification / post-embryonic development / maturation of LSU-rRNA / ribosomal large subunit biogenesis / regulation of signal transduction by p53 class mediator / innate immune response in mucosa / skeletal system development / mRNA 3'-UTR binding / positive regulation of translation / calcium ion transmembrane transport / sensory perception of sound / calcium channel activity / bone development / ribosomal large subunit assembly / cellular response to gamma radiation / mRNA 5'-UTR binding / Regulation of expression of SLITs and ROBOs / cytoplasmic ribonucleoprotein granule / cellular response to type II interferon / intracellular calcium ion homeostasis Similarity search - Function
Calcium load-activated calcium channel / PAT complex subunit CCDC47 / Nicalin / Transmembrane protein 147 / PAT complex subunit CCDC47 / Predicted membrane protein (DUF2053) / Integral membrane protein EMC3/TMCO1-like / Integral membrane protein EMC3/TMCO1-like / Integral membrane protein DUF106 / Protein transport Sec61-beta/Sbh ...Calcium load-activated calcium channel / PAT complex subunit CCDC47 / Nicalin / Transmembrane protein 147 / PAT complex subunit CCDC47 / Predicted membrane protein (DUF2053) / Integral membrane protein EMC3/TMCO1-like / Integral membrane protein EMC3/TMCO1-like / Integral membrane protein DUF106 / Protein transport Sec61-beta/Sbh / Protein transport protein SecG/Sec61-beta/Sbh / Sec61beta family / Protein translocase SEC61 complex, gamma subunit / Protein translocase SecE domain superfamily / Translocon Sec61/SecY, plug domain / Plug domain of Sec61p / Protein secE/sec61-gamma signature. / Protein secY signature 1. / Protein secY signature 2. / SecE/Sec61-gamma subunits of protein translocation complex / Protein translocase complex, SecE/Sec61-gamma subunit / SecY/SEC61-alpha family / SecY domain superfamily / SecY conserved site / SecY / Peptidase M28 / Peptidase family M28 / Ribosomal protein L6, N-terminal / Ribosomal protein L6, N-terminal domain / Ribosomal protein L30e / Ribosomal protein L2, archaeal-type / Ribosomal protein L28e / Ribosomal L15/L27a, N-terminal / Ribosomal protein L23 / Ribosomal L28e/Mak16 / Ribosomal L28e protein family / Ribosomal protein L41 / Ribosomal protein L41 / metallochaperone-like domain / TRASH domain / Ribosomal protein L29e / Ribosomal L29e protein family / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L22e protein family / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal L38e protein family / Ribosomal protein L27e, conserved site / Ribosomal protein L27e signature. / Ribosomal protein L44e signature. / Ribosomal protein L10e, conserved site / Ribosomal protein L10e signature. / Ribosomal protein L10e / Ribosomal protein L19, eukaryotic / Ribosomal protein L13e / Ribosomal protein L13e / 60S ribosomal protein L18a/ L20, eukaryotes / : / Ribosomal protein L19/L19e conserved site / Ribosomal protein L19e signature. / Ribosomal protein L24e, conserved site / Ribosomal protein L24e signature. / Ribosomal protein L44e / Ribosomal protein L44 / Ribosomal protein L34e, conserved site / Ribosomal protein L34e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / Ribosomal protein L6e signature. / 50S ribosomal protein L18Ae/60S ribosomal protein L20 and L18a / Ribosomal protein L30e signature 1. / Ribosomal protein 50S-L18Ae/60S-L20/60S-L18A / Ribosomal proteins 50S-L18Ae/60S-L20/60S-L18A / Ribosomal protein L23/L25, N-terminal / Ribosomal protein L23, N-terminal domain / Eukaryotic Ribosomal Protein L27, KOW domain / Ribosomal protein L30e signature 2. / Ribosomal protein L30e, conserved site / Ribosomal protein 60S L18 and 50S L18e / Ribosomal protein L27e / Ribosomal protein L27e superfamily / Ribosomal L27e protein family / Ribosomal protein L36e signature. / Ribosomal protein L39e, conserved site / Ribosomal protein L39e signature. / 60S ribosomal protein L19 / Ribosomal protein L34Ae / Ribosomal protein L34e / 60S ribosomal protein L35 / Ribosomal protein L35Ae, conserved site / Ribosomal protein L35Ae signature. / Ribosomal protein L18/L18-A/B/e, conserved site / Ribosomal Protein L6, KOW domain / Ribosomal protein L18e signature. / Ribosomal protein L30/YlxQ / Ribosomal protein L7A/L8 / Ribosomal protein L6e Similarity search - Domain/homology
Large ribosomal subunit protein eL42 / Large ribosomal subunit protein eL33 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein eL13 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein eL22 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL3 ...Large ribosomal subunit protein eL42 / Large ribosomal subunit protein eL33 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein eL13 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein eL22 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein eL21 / Large ribosomal subunit protein eL28 / Large ribosomal subunit protein eL29 / Large ribosomal subunit protein eL34 / Large ribosomal subunit protein eL14 / Protein transport protein Sec61 subunit gamma / Protein transport protein Sec61 subunit beta / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein eL15 / Large ribosomal subunit protein eL27 / Large ribosomal subunit protein eL43 / Protein transport protein Sec61 subunit alpha isoform 1 / Large ribosomal subunit protein eL37 / Large ribosomal subunit protein eL8 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein eL32 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein uL2 / Small ribosomal subunit protein eS32 / Large ribosomal subunit protein eL38 / Large ribosomal subunit protein eL24 / Large ribosomal subunit protein eL19 / Large ribosomal subunit protein eL20 / Large ribosomal subunit protein eL6 / Large ribosomal subunit protein eL18 / Nicalin / Coiled-coil domain-containing protein 47 / Transmembrane protein 147 / Calcium load-activated calcium channel / Large ribosomal subunit protein eL36 Similarity search - Component
Biological species
Homo sapiens (human)
Method
single particle reconstruction / cryo EM / Resolution: 3.8 Å
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1R01GM130051-01
United States
Citation
Journal: Elife / Year: 2020 Title: An ER translocon for multi-pass membrane protein biogenesis. Authors: Philip T McGilvray / S Andrei Anghel / Arunkumar Sundaram / Frank Zhong / Michael J Trnka / James R Fuller / Hong Hu / Alma L Burlingame / Robert J Keenan / Abstract: Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here ...Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.
History
Deposition
Feb 21, 2020
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Header (metadata) release
Mar 25, 2020
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Map release
Sep 2, 2020
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Update
Sep 2, 2020
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Current status
Sep 2, 2020
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : Native complex of a translating ribosome and a novel translocon c...
Entire
Name: Native complex of a translating ribosome and a novel translocon containing the Sec61 complex, TMEM147, NOMO, Nicalin, CCDC47, and TMCO1.
Components
Complex: Native complex of a translating ribosome and a novel translocon containing the Sec61 complex, TMEM147, NOMO, Nicalin, CCDC47, and TMCO1.
Complex: 60S Subunit
Complex: 60S acidic ribosomal protein P0
Complex: 60S acidic ribosomal protein P1
Complex: 60S acidic ribosomal protein P2
Complex: 60S ribosomal protein L10
Complex: 60S ribosomal protein L10a
Complex: 60S ribosomal protein L11
Complex: 60S ribosomal protein L12
Complex: 60S ribosomal protein L13
Complex: 60S ribosomal protein L13a
Complex: 60S ribosomal protein L14
Complex: 60S ribosomal protein L15
Complex: 60S ribosomal protein L17
Complex: 60S ribosomal protein L18
Complex: 60S ribosomal protein L18a
Complex: 60S ribosomal protein L19
Complex: 60S ribosomal protein L21
Complex: 60S ribosomal protein L22
Complex: 60S ribosomal protein L23
Complex: 60S ribosomal protein L23a
Complex: 60S ribosomal protein L24
Complex: 60S ribosomal protein L26
Complex: 60S ribosomal protein L27
Complex: 60S ribosomal protein L27a
Complex: 60S ribosomal protein L28
Complex: 60S ribosomal protein L3
Complex: 60S ribosomal protein L30
Complex: 60S ribosomal protein L31
Complex: 60S ribosomal protein L32
Complex: 60S ribosomal protein L34
Complex: 60S ribosomal protein L35
Complex: 60S ribosomal protein L35a
Complex: 60S ribosomal protein L36
Complex: 60S ribosomal protein L36a
Complex: 60S ribosomal protein L37a
Complex: 60S ribosomal protein L38
Complex: 60S ribosomal protein L39
Complex: 60S ribosomal protein L4
Complex: 60S ribosomal protein L5
Complex: 60S ribosomal protein L6
Complex: 60S ribosomal protein L7
Complex: 60S ribosomal protein L7a
Complex: 60S ribosomal protein L8
Complex: 60S ribosomal protein L9
Complex: 40S Subunit
Complex: 40S ribosomal protein S10
Complex: 40S ribosomal protein S11
Complex: 40S ribosomal protein S12
Complex: 40S ribosomal protein S13
Complex: 40S ribosomal protein S14
Complex: 40S ribosomal protein S15
Complex: 40S ribosomal protein S15a
Complex: 40S ribosomal protein S16
Complex: 40S ribosomal protein S17
Complex: 40S ribosomal protein S18
Complex: 40S ribosomal protein S19
Complex: 40S ribosomal protein S2
Complex: 40S ribosomal protein S20
Complex: 40S ribosomal protein S21
Complex: 40S ribosomal protein S23
Complex: 40S ribosomal protein S24
Complex: 40S ribosomal protein S25
Complex: 40S ribosomal protein S26
Complex: 40S ribosomal protein S27
Complex: 40S ribosomal protein S27-like
Complex: 40S ribosomal protein S28
Complex: 40S ribosomal protein S29
Complex: 40S ribosomal protein S3
Complex: 40S ribosomal protein S30
Complex: 40S ribosomal protein S3a
Complex: 40S ribosomal protein S4, X isoform
Complex: 40S ribosomal protein S5
Complex: 40S ribosomal protein S6
Complex: 40S ribosomal protein S7
Complex: 40S ribosomal protein S8
Complex: 40S ribosomal protein S9
Complex: 40S ribosomal protein SA
Complex: Translocon
Complex: Sec61 Complex
Complex: TMCO1
Complex: NOMO-Nicalin-TMEM147 Complex
Complex: CCDC47
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Supramolecule #1: Native complex of a translating ribosome and a novel translocon c...
Supramolecule
Name: Native complex of a translating ribosome and a novel translocon containing the Sec61 complex, TMEM147, NOMO, Nicalin, CCDC47, and TMCO1. type: complex / ID: 1 / Parent: 0
Source (natural)
Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum
Molecular weight
Theoretical: 3.92 MDa
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Supramolecule #2: 60S Subunit
Supramolecule
Name: 60S Subunit / type: complex / ID: 2 / Parent: 1 Details: 60S ribosomal protein L9 60S ribosomal protein L8 60S ribosomal protein L7a 60S ribosomal protein L7 60S ribosomal protein L6 60S ribosomal protein L5 60S ribosomal protein L4 60S ribosomal ...Details: 60S ribosomal protein L9 60S ribosomal protein L8 60S ribosomal protein L7a 60S ribosomal protein L7 60S ribosomal protein L6 60S ribosomal protein L5 60S ribosomal protein L4 60S ribosomal protein L39 60S ribosomal protein L38 60S ribosomal protein L37a 60S ribosomal protein L36a 60S ribosomal protein L36 60S ribosomal protein L35a 60S ribosomal protein L35 60S ribosomal protein L34 60S ribosomal protein L32 60S ribosomal protein L31 60S ribosomal protein L30 60S ribosomal protein L3 60S ribosomal protein L28 60S ribosomal protein L27a 60S ribosomal protein L27 60S ribosomal protein L26 60S ribosomal protein L24 60S ribosomal protein L23a 60S ribosomal protein L23 60S ribosomal protein L22-like 1 60S ribosomal protein L22 60S ribosomal protein L21 60S ribosomal protein L19 60S ribosomal protein L18a 60S ribosomal protein L18 60S ribosomal protein L17 60S ribosomal protein L15 60S ribosomal protein L14 60S ribosomal protein L13a 60S ribosomal protein L13 60S ribosomal protein L12 60S ribosomal protein L11 60S ribosomal protein L10a 60S ribosomal protein L10 60S ribosomal export protein NMD3 60S acidic ribosomal protein P2 60S acidic ribosomal protein P1 60S acidic ribosomal protein P0-like 60S acidic ribosomal protein P0 Ubiquitin-60S ribosomal protein L40
Source (natural)
Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum
Molecular weight
Theoretical: 2.42 MDa
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Supramolecule #3: 40S Subunit
Supramolecule
Name: 40S Subunit / type: complex / ID: 3 / Parent: 1 Details: 40S ribosomal protein SA 40S ribosomal protein S9 40S ribosomal protein S8 40S ribosomal protein S7 40S ribosomal protein S6 40S ribosomal protein S5 40S ribosomal protein S4, X isoform 40S ...Details: 40S ribosomal protein SA 40S ribosomal protein S9 40S ribosomal protein S8 40S ribosomal protein S7 40S ribosomal protein S6 40S ribosomal protein S5 40S ribosomal protein S4, X isoform 40S ribosomal protein S3a 40S ribosomal protein S30 40S ribosomal protein S3 40S ribosomal protein S29 40S ribosomal protein S28 40S ribosomal protein S27-like 40S ribosomal protein S27 40S ribosomal protein S26 40S ribosomal protein S25 40S ribosomal protein S24 40S ribosomal protein S23 40S ribosomal protein S21 40S ribosomal protein S20 40S ribosomal protein S2 40S ribosomal protein S19 40S ribosomal protein S18 40S ribosomal protein S17 40S ribosomal protein S16 40S ribosomal protein S15a 40S ribosomal protein S15 40S ribosomal protein S14 40S ribosomal protein S13 40S ribosomal protein S12 40S ribosomal protein S11 40S ribosomal protein S10 Ubiquitin-40S ribosomal protein S27a
Source (natural)
Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum
Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum
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Supramolecule #83: 40S ribosomal protein SA
Supramolecule
Name: 40S ribosomal protein SA / type: complex / ID: 83 / Parent: 3
Source (natural)
Organism: Homo sapiens (human) / Organ: Kidney / Cell: HEK293 / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
0.4 mg/mL
Buffer
pH: 7.4 Component:
Concentration
Formula
Name
150.0 mM
C2H3KO2
potassium acetate
50.0 mM
C8H18N2O4S
HEPES, pH 7.4
5.0 mM
MgCl2
magnesium chloride
0.25 percent (w/v)
C56H92O29
digitonin
Grid
Model: C-flat-1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE / Details: unspecified
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV Details: Two filter papers were added to each arm, 2.5 microliters of sample were added to grids, and 0.5 second of drain time was allowed before vitrification..
Details
Sample was well-dispersed on a thin (~2nm) carbon film.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 (6k x 4k) / #0 - Detector mode: COUNTING / #0 - Number grids imaged: 1 / #0 - Number real images: 5562 / #0 - Average exposure time: 3.8 sec. / #0 - Average electron dose: 50.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 (6k x 4k) / #1 - Number grids imaged: 1 / #1 - Number real images: 5562 / #1 - Average exposure time: 3.8 sec. / #1 - Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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