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- PDB-6zzz: Crystal structure of yeast Sec62 cytoplasmic domain -

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Basic information

Entry
Database: PDB / ID: 6zzz
TitleCrystal structure of yeast Sec62 cytoplasmic domain
ComponentsTranslocation protein SEC62
KeywordsPROTEIN TRANSPORT / Sec62 / Sec62 domain / Post translocon
Function / homology
Function and homology information


Sec62/Sec63 complex / translocon complex / rough endoplasmic reticulum membrane / post-translational protein targeting to membrane, translocation / protein transmembrane transporter activity / endoplasmic reticulum / membrane
Similarity search - Function
Translocation protein Sec62, ascomycota / Translocation protein Sec62 / Translocation protein Sec62
Similarity search - Domain/homology
Translocation protein SEC62
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.54 Å
AuthorsCheng, J. / Beckmann, R.
CitationJournal: EMBO J / Year: 2021
Title: Architecture of the active post-translational Sec translocon.
Authors: Tsai-Hsuan Weng / Wieland Steinchen / Birgitta Beatrix / Otto Berninghausen / Thomas Becker / Gert Bange / Jingdong Cheng / Roland Beckmann /
Abstract: In eukaryotes, most secretory and membrane proteins are targeted by an N-terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein-conducting channel ...In eukaryotes, most secretory and membrane proteins are targeted by an N-terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein-conducting channel (PCC). In the post-translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre-opened state. Here, we present a cryo-EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N-terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C-terminus of Sec63. Together, we obtained a near-complete and integrated model of the active Sec complex.
History
DepositionAug 5, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 2, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 30, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 10, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Translocation protein SEC62
B: Translocation protein SEC62
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,16120
Polymers30,3412
Non-polymers1,82018
Water1,36976
1
A: Translocation protein SEC62
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,21912
Polymers15,1701
Non-polymers1,04911
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Translocation protein SEC62
hetero molecules


Theoretical massNumber of molelcules
Total (without water)15,9428
Polymers15,1701
Non-polymers7727
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)99.206, 99.206, 155.657
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522
Space group name HallP652(x,y,z+1/12)
Symmetry operation#1: x,y,z
#2: x-y,x,z+5/6
#3: y,-x+y,z+1/6
#4: -y,x-y,z+2/3
#5: -x+y,-x,z+1/3
#6: x-y,-y,-z
#7: -x,-x+y,-z+1/3
#8: -x,-y,z+1/2
#9: y,x,-z+2/3
#10: -y,-x,-z+1/6
#11: -x+y,y,-z+1/2
#12: x,x-y,-z+5/6
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain 'A'A18 - 96
121chain 'A'A99 - 143
231(chain 'B' and (resid 18 through 118 or (resid 119...B18 - 96
241(chain 'B' and (resid 18 through 118 or (resid 119...B99 - 143

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Components

#1: Protein Translocation protein SEC62 / Sec62/63 complex 30 kDa subunit


Mass: 15170.269 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SEC62, YPL094C, LPG14C / Production host: Escherichia coli (E. coli) / References: UniProt: P21825
#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID / MES (buffer)


Mass: 195.237 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 76 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.64 Å3/Da / Density % sol: 66.25 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / Details: 100mM MES pH 5.7-5.9, 2 M (NH4)2SO4 / PH range: 5.7-5.9

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM02 / Wavelength: 0.97958 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: May 19, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97958 Å / Relative weight: 1
ReflectionResolution: 2.54→47.26 Å / Num. obs: 15563 / % possible obs: 99.81 % / Redundancy: 38.1 % / Biso Wilson estimate: 74.11 Å2 / CC1/2: 1 / CC star: 1 / Rmerge(I) obs: 0.1075 / Rpim(I) all: 0.01778 / Rrim(I) all: 0.109 / Net I/σ(I): 26.56
Reflection shellResolution: 2.54→2.63 Å / Rmerge(I) obs: 1.869 / Mean I/σ(I) obs: 2.03 / Num. unique obs: 1495 / CC1/2: 0.949 / Rrim(I) all: 1.894

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
XDSdata reduction
XDSdata scaling
PHENIXphasing
RefinementMethod to determine structure: SAD / Resolution: 2.54→47.26 Å / SU ML: 0.3284 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 27.2985
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2598 778 5 %
Rwork0.2206 14785 -
obs0.2225 15563 99.68 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 75.34 Å2
Refinement stepCycle: LAST / Resolution: 2.54→47.26 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2108 0 99 76 2283
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01282240
X-RAY DIFFRACTIONf_angle_d1.7293022
X-RAY DIFFRACTIONf_chiral_restr0.0631308
X-RAY DIFFRACTIONf_plane_restr0.0094376
X-RAY DIFFRACTIONf_dihedral_angle_d23.5407289
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.54-2.70.35941250.28032378X-RAY DIFFRACTION98.54
2.7-2.910.32681250.26912401X-RAY DIFFRACTION99.92
2.91-3.20.29771280.25162438X-RAY DIFFRACTION100
3.2-3.660.25531290.22352435X-RAY DIFFRACTION99.96
3.66-4.610.2391310.19382486X-RAY DIFFRACTION99.96
4.61-47.260.2491400.21822647X-RAY DIFFRACTION99.68

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