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- PDB-6v05: Cryo-EM structure of a substrate-engaged Bam complex -

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Basic information

Entry
Database: PDB / ID: 6v05
TitleCryo-EM structure of a substrate-engaged Bam complex
Components(Outer membrane protein assembly factor ...) x 6
KeywordsMEMBRANE PROTEIN / beta-barrel / insertase
Function / homology
Function and homology information


Bam protein complex / Gram-negative-bacterium-type cell outer membrane assembly / protein insertion into membrane / cell outer membrane / protein-macromolecule adaptor activity / cell adhesion / response to antibiotic / cell surface / membrane / identical protein binding
Similarity search - Function
PQQ enzyme repeat / Outer membrane protein assembly factor BamB / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamC / Outer membrane protein assembly factor BamC, C-terminal / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamE / Lipoprotein SmpA/OmlA / Outer membrane lipoprotein BamD-like ...PQQ enzyme repeat / Outer membrane protein assembly factor BamB / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamC / Outer membrane protein assembly factor BamC, C-terminal / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamE / Lipoprotein SmpA/OmlA / Outer membrane lipoprotein BamD-like / SmpA / OmlA family / Outer membrane lipoprotein / BamE-like / Outer membrane protein assembly factor BamA / Pyrrolo-quinoline quinone repeat / PQQ-like domain / POTRA domain, BamA/TamA-like / Surface antigen variable number repeat / POTRA domain / POTRA domain profile. / Pyrrolo-quinoline quinone beta-propeller repeat / beta-propeller repeat / Surface antigen D15-like / Bacterial surface antigen (D15) / Omp85 superfamily domain / Quinoprotein alcohol dehydrogenase-like superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile. / Tetratricopeptide-like helical domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamE / Outer membrane protein assembly factor BamC / Outer membrane protein assembly factor BamE / Outer membrane protein assembly factor BamA / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamB / Outer membrane protein assembly factor BamC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsTomasek, D. / Rawson, S. / Lee, J. / Wzorek, J.S. / Harrison, S.C. / Li, Z. / Kahne, D.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI081059 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F31GM116210 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM108258 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2020
Title: Structure of a nascent membrane protein as it folds on the BAM complex.
Authors: David Tomasek / Shaun Rawson / James Lee / Joseph S Wzorek / Stephen C Harrison / Zongli Li / Daniel Kahne /
Abstract: Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure. β-Barrels are sheets of β-strands ...Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure. β-Barrels are sheets of β-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane. One subunit of the machines is itself a β-barrel protein that has a central role in folding other β-barrels. In Gram-negative bacteria, the β-barrel assembly machine (BAM) consists of the β-barrel protein BamA, and four lipoproteins. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP), we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid β-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.
History
DepositionNov 18, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2020Provider: repository / Type: Initial release
Revision 1.1Jun 24, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 29, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Outer membrane protein assembly factor BamA
B: Outer membrane protein assembly factor BamB
C: Outer membrane protein assembly factor BamC
D: Outer membrane protein assembly factor BamD
E: Outer membrane protein assembly factor BamE
F: Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA


Theoretical massNumber of molelcules
Total (without water)274,7606
Polymers274,7606
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Outer membrane protein assembly factor ... , 6 types, 6 molecules ABCDEF

#1: Protein Outer membrane protein assembly factor BamA / Omp85


Mass: 90567.227 Da / Num. of mol.: 1 / Mutation: C690S, C700S, F804C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: bamA, yaeT, yzzN, yzzY, b0177, JW0172 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A940
#2: Protein Outer membrane protein assembly factor BamB


Mass: 41918.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: bamB, yfgL, b2512, JW2496 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P77774
#3: Protein Outer membrane protein assembly factor BamC


Mass: 36875.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12
Gene: bamC, nlpB, A6581_02455, A6592_19755, A8C65_04235, A8G17_10975, AC067_02730, AC789_1c28090, ACN002_2512, ACN68_11560, ACN81_26800, ACU57_04515, ACU90_01100, AKG99_13055, AM270_06735, AM446_ ...Gene: bamC, nlpB, A6581_02455, A6592_19755, A8C65_04235, A8G17_10975, AC067_02730, AC789_1c28090, ACN002_2512, ACN68_11560, ACN81_26800, ACU57_04515, ACU90_01100, AKG99_13055, AM270_06735, AM446_08445, AM464_03885, AML07_10810, AML35_21815, APT94_12605, APZ14_05885, AUQ13_12835, AUS26_08895, AW106_01925, AWP75_05795, B1K96_18050, B9M99_18180, B9T59_21405, BANRA_01021, BANRA_03170, BANRA_04601, BB545_17690, BE963_28130, BEN53_15545, BER14_02350, BHF46_00865, BHS81_15170, BIQ87_14450, BIU72_15910, BIZ41_13405, BK248_16985, BK292_09100, BK373_06140, BK375_00595, BK383_22560, BK400_08740, BMT91_12040, BOH76_02570, BON66_22990, BON69_13590, BON71_05810, BON75_17940, BON76_02345, BON81_08015, BON83_11105, BON86_12870, BON92_23095, BON94_05640, BON95_12010, BON96_16290, BTQ06_24710, BUE81_00175, BvCms2454_00576, BvCms28BK_03651, BvCmsA75A_03498, BvCmsC61A_03920, BvCmsHHP001_02989, BvCmsHHP019_02257, BvCmsHHP056_03727, BvCmsKKP061_00365, BvCmsKSNP019_02678, BvCmsKSNP073_01370, BvCmsKSNP081_01804, BvCmsKSNP120_01449, BvCmsKSP011_03652, BvCmsKSP024_03770, BvCmsKSP026_00127, BvCmsKSP045_01057, BvCmsKSP058_05104, BvCmsKSP067_01143, BvCmsNSNP036_03233, BvCmsNSP007_05045, BvCmsNSP047_01191, BvCmsNSP072_03470, BvCmsSINP011_00589, BvCmsSINP012_02461, BvCmsSINP022_03246, BVL39_14450, BW690_08400, BXT93_09065, BZL31_19830, BZL69_23975, C3449_00575, C4J69_10905, C5N07_10515, C5P01_06205, C5P43_23745, C5P44_22155, C6669_16365, C6986_16055, C7235_07205, C9025_02075, C9083_01895, C9212_02290, C9299_09010, C9E25_13305, C9Y80_06105, C9Y95_21475, C9Z12_16640, CA593_14465, CDL37_24220, CEG98_13220, CG692_24660, CI641_005275, CI694_19990, CIJ94_07570, COD30_00650, COD46_27330, CQP61_08535, CR538_07295, CRM83_27895, CRT46_14950, CWM24_12580, CWS33_10415, CY655_15855, D2184_13985, D2188_18170, D3821_11830, D3822_05395, D3O91_03970, D3Y67_12985, D9D31_00585, D9D43_09275, D9E34_00585, D9E35_17030, D9F17_05285, D9G42_10275, D9G48_19890, D9H68_00610, D9H70_20745, D9H94_18900, D9I11_00510, D9I18_08285, D9I87_03080, D9I88_00655, D9J11_04670, D9J44_00445, D9J60_12530, D9K48_26220, D9K54_08830, DAH27_02200, DAH30_02900, DAH34_02675, DAH37_19960, DBQ99_07990, DD762_13530, DEN86_22815, DEN89_10395, DEN97_02085, DEO19_00630, DIV22_31745, DL545_07645, DL800_18795, DM102_07120, DM129_09435, DNQ41_17525, DNQ45_02690, DP258_07610, DQE83_18935, DQF57_11025, DQO13_11190, DS732_18480, DTL43_01510, DTL90_08635, DTM10_01015, DTM25_19255, DTM45_09650, DU321_10720, DWB25_07365, DXT71_21935, E2112_00485, E2115_16230, E2119_05135, E2126_07380, E2127_04515, E2129_06730, E2132_10070, E2134_09470, E2135_00555, E2855_03225, E2863_03132, E5S46_08270, E5S47_00585, E5S58_10625, E5S61_11100, EAI42_12280, EAI52_02570, EC1094V2_1211, EC3234A_44c00830, EC382_03360, EC95NR1_01696, ECTO6_01409, ED600_00620, ED648_19505, EEP23_23015, EHH55_03705, EIA21_06830, EJC75_11395, EKI52_22355, EL75_1174, EL79_1185, EL80_1190, ELT58_05595, ELU85_09985, ELV08_03665, ELV26_03145, ELV28_11920, EO240_05615, EO241_29005, EPS71_00265, EPS97_10555, EPT01_15160, EQ820_10645, EQ823_08955, EQ825_24025, EQ830_05480, ERL57_11390, ERS085365_02853, ERS085366_00345, ERS085374_00117, ERS085379_00986, ERS085383_02107, ERS085386_00039, ERS085404_01131, ERS085416_01322, ERS139211_02639, ERS150876_00778, ExPECSC038_04470, EXX06_10435, EXX13_08605, EXX23_06350, EXX40_08105, EXX53_08115, EXX55_10135, EXX71_05165, EXX73_17945, EXX78_07240, EXX87_17745, EYD11_06585, EYY78_21115, FAX15_16530, FE198_07360, FNJ69_10855, FNJ79_09165, FNJ83_16390, FORC28_1482, FQR64_06705, FV293_04230, HmCms184_03495, HmCmsJML072_03247, HmCmsJML074_00064, HmCmsJML204_03962, HMPREF3040_02807, HW43_16770, JD73_18025, MJ49_18500, NCTC10090_04434, NCTC10764_00647, NCTC10865_01743, NCTC11022_02534, NCTC11126_06126, NCTC11181_03634, NCTC11341_00816, NCTC12950_01648, NCTC13148_02274, NCTC13462_05111, NCTC13846_01494, NCTC7927_01568, NCTC8621_01448, NCTC8960_04114, NCTC9036_01478, NCTC9044_00198, NCTC9045_01669, NCTC9050_04702, NCTC9055_03350, NCTC9062_01737, NCTC9077_01815, NCTC9111_01883, NCTC9117_01960, NCTC9119_01583, NCTC9702_01656, NCTC9703_01031, NCTC9706_04674, NCTC9969_01610, PGD_00767, RG28_09510, RK56_024525, RX35_01364, SAMEA3472033_00601, SAMEA3472043_00977, SAMEA3472044_02057, SAMEA3472047_03903, SAMEA3472055_00995, SAMEA3472056_00816, SAMEA3472070_00123, SAMEA3472080_01551, SAMEA3472090_01290, SAMEA3472108_03434, SAMEA3472147_00102, SAMEA3484427_00337, SAMEA3484429_00446, SAMEA3484434_00350, SAMEA3485101_04932, SAMEA3485113_01786, SAMEA3752557_01026, SAMEA3752559_00799, SAMEA3752620_00108, SAMEA3753097_02061, SAMEA3753164_00674, SAMEA3753300_00970, SK85_02726, UC41_17115, UN86_00295, UN91_14105, WQ89_05460, WR15_19465, YDC107_1000
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: W8SZY2, UniProt: P0A903*PLUS
#4: Protein Outer membrane protein assembly factor BamD


Mass: 27858.350 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: yfiO, bamD, ECIAI39_2800 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0H3MJ38, UniProt: P0AC02*PLUS
#5: Protein Outer membrane protein assembly factor BamE


Mass: 13530.256 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: smpA, bamE, NCTC8196_03449 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A3S5E0E0, UniProt: P0A937*PLUS
#6: Protein Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA,Outer membrane protein assembly factor BamA / Omp85


Mass: 64010.285 Da / Num. of mol.: 1
Mutation: T467C,C690S,C700S,deletion of residues 172-421,deletion of residues 430-439
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: bamA, yaeT, yzzN, yzzY, b0177, JW0172 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A940

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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: BamABCDE bound to substrate BamA with loop 1 deleted / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethane hydrochlorideTris-HClTris1
2150 mMsodium chlorideNaClSodium chloride1
30.02 %glyco-diosgeninGDN1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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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: OTHER
Electron lensMode: OTHER / Calibrated magnification: 58717 X / Calibrated defocus min: 1100 nm / Calibrated defocus max: 2800 nm
Image recordingAverage exposure time: 3 sec. / Electron dose: 70 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 4097
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 25 eV

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Processing

EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2054956
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 223353 / Symmetry type: POINT

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