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- PDB-8bvj: Hfq-Crc-estA translation repression complex -

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Basic information

Entry
Database: PDB / ID: 8bvj
TitleHfq-Crc-estA translation repression complex
Components
  • Catabolite repression control protein
  • RNA-binding protein Hfq
  • estA mRNA
KeywordsRNA BINDING PROTEIN / co-transcriptional RNA folding / Crc / metabolic regulation / ribonucleoprotein assembly / RNA chaperone Hfq / translational regulation
Function / homology
Function and homology information


double-stranded DNA 3'-5' DNA exonuclease activity / exodeoxyribonuclease III / DNA repair / regulation of DNA-templated transcription / RNA binding / metal ion binding
Similarity search - Function
Exodeoxyribonuclease III-like / RNA-binding protein Hfq / Hfq protein / AP endonuclease 1 / AP endonucleases family 1 profile. / Endonuclease/exonuclease/phosphatase / Endonuclease/Exonuclease/phosphatase family / : / Sm domain profile. / Endonuclease/exonuclease/phosphatase superfamily / LSM domain superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / RNA-binding protein Hfq / Catabolite repression control protein
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsDendooven, T. / Luisi, B.F.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: EMBO J / Year: 2023
Title: Translational regulation by Hfq-Crc assemblies emerges from polymorphic ribonucleoprotein folding.
Authors: Tom Dendooven / Elisabeth Sonnleitner / Udo Bläsi / Ben F Luisi /
Abstract: The widely occurring bacterial RNA chaperone Hfq is a key factor in the post-transcriptional control of hundreds of genes in Pseudomonas aeruginosa. How this broadly acting protein can contribute to ...The widely occurring bacterial RNA chaperone Hfq is a key factor in the post-transcriptional control of hundreds of genes in Pseudomonas aeruginosa. How this broadly acting protein can contribute to the regulatory requirements of many different genes remains puzzling. Here, we describe cryo-EM structures of higher order assemblies formed by Hfq and its partner protein Crc on control regions of different P. aeruginosa target mRNAs. Our results show that these assemblies have mRNA-specific quaternary architectures resulting from the combination of multivalent protein-protein interfaces and recognition of patterns in the RNA sequence. The structural polymorphism of these ribonucleoprotein assemblies enables selective translational repression of many different target mRNAs. This system elucidates how highly complex regulatory pathways can evolve with a minimal economy of proteinogenic components in combination with RNA sequence and fold.
History
DepositionDec 4, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: RNA-binding protein Hfq
T: RNA-binding protein Hfq
J: RNA-binding protein Hfq
L: RNA-binding protein Hfq
N: RNA-binding protein Hfq
P: RNA-binding protein Hfq
X: RNA-binding protein Hfq
Y: RNA-binding protein Hfq
S: RNA-binding protein Hfq
U: RNA-binding protein Hfq
V: RNA-binding protein Hfq
W: RNA-binding protein Hfq
K: RNA-binding protein Hfq
M: RNA-binding protein Hfq
D: RNA-binding protein Hfq
E: RNA-binding protein Hfq
F: RNA-binding protein Hfq
I: RNA-binding protein Hfq
Q: Catabolite repression control protein
B: estA mRNA
G: Catabolite repression control protein
C: Catabolite repression control protein
A: Catabolite repression control protein


Theoretical massNumber of molelcules
Total (without water)322,46623
Polymers322,46623
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47190 Å2
ΔGint-250 kcal/mol
Surface area101040 Å2

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Components

#1: Protein
RNA-binding protein Hfq


Mass: 9114.487 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: hfq, PSPA7_5673 / Production host: Escherichia coli (E. coli) / References: UniProt: A6VD57
#2: Protein
Catabolite repression control protein / Exodeoxyribonuclease III


Mass: 30101.869 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria)
Gene: crc, exoA, ALP65_01013, CAZ10_32350, CGU42_22755, DT376_09125, E4V10_18585, E5D53_27785, E5Z62_16095, E5Z63_23405, ECC04_031800, GNQ48_25180, GUL26_21635, IPC111_12165, IPC112_13745, IPC113_ ...Gene: crc, exoA, ALP65_01013, CAZ10_32350, CGU42_22755, DT376_09125, E4V10_18585, E5D53_27785, E5Z62_16095, E5Z63_23405, ECC04_031800, GNQ48_25180, GUL26_21635, IPC111_12165, IPC112_13745, IPC113_22800, IPC114_17225, IPC115_20105, IPC116_23795, IPC117_25625, IPC118_26680, IPC119_29495, IPC120_26430, IPC121_21355, IPC122_22810, IPC123_28525, IPC124_13695, IPC125_17530, IPC126_13890, IPC127_14855, IPC128_10355, IPC1295_10410, IPC129_12605, IPC1306_08360, IPC1307_28280, IPC1308_08250, IPC1309_21675, IPC130_13760, IPC1310_18070, IPC1311_27715, IPC1312_27760, IPC1313_12995, IPC1314_23305, IPC1315_13925, IPC1316_03820, IPC1317_21325, IPC1319_28440, IPC131_02850, IPC1320_21925, IPC1321_13670, IPC1322_08030, IPC1323_08650, IPC1324_08655, IPC1325_28715, IPC1326_03390, IPC1327_03390, IPC1329_17010, IPC132_06970, IPC1330_11465, IPC1331_28200, IPC1332_29425, IPC1333_11845, IPC1334_25345, IPC1335_28860, IPC1336_09225, IPC1337_29545, IPC1339_28790, IPC133_03360, IPC1340_28180, IPC1341_12600, IPC1342_25815, IPC1343_28125, IPC1345_26635, IPC1346_12480, IPC1347_06540, IPC1349_02495, IPC134_02845, IPC135_03360, IPC137_05170, IPC139_09290, IPC140_06730, IPC141_03365, IPC142_07260, IPC143_27785, IPC144_27850, IPC145_26565, IPC146_07875, IPC1474_29365, IPC1476_04745, IPC1477_00205, IPC1478_21530, IPC1479_05830, IPC147_27530, IPC1480_09320, IPC1481_08100, IPC1482_03390, IPC1485_09070, IPC1486_00200, IPC1487_09730, IPC1488_23420, IPC1489_30180, IPC148_13460, IPC1490_30060, IPC1491_03590, IPC1492_29195, IPC1494_10055, IPC1496_07955, IPC1498_13815, IPC1499_04620, IPC149_07870, IPC1500_13950, IPC1501_16685, IPC1502_17430, IPC1503_12005, IPC1504_12415, IPC1505_29825, IPC1506_17410, IPC1507_24155, IPC1508_30170, IPC1509_28695, IPC150_28080, IPC1510_28915, IPC1511_30420, IPC1512_29660, IPC1515_25380, IPC1516_25030, IPC1517_08725, IPC1518_07355, IPC1519_08150, IPC151_23655, IPC1521_08375, IPC1522_08210, IPC1523_29925, IPC152_28760, IPC153_23345, IPC154_17265, IPC155_18160, IPC156_17280, IPC157_20765, IPC1583_04500, IPC1584_21700, IPC1585_03355, IPC1586_12135, IPC1587_07630, IPC1588_00205, IPC1589_12850, IPC158_22475, IPC1590_04505, IPC1591_26650, IPC1592_03360, IPC1593_28620, IPC1594_08340, IPC1595_28610, IPC1596_08050, IPC1597_07705, IPC1598_07535, IPC1599_12520, IPC159_19675, IPC1600_03625, IPC1601_25405, IPC1602_28860, IPC1603_04480, IPC1604_17280, IPC1605_06980, IPC1606_28085, IPC161_21475, IPC168_27215, IPC172_03365, IPC173_03365, IPC174_04115, IPC175_27990, IPC176_00200, IPC177_23820, IPC178_12645, IPC179_03225, IPC180_03440, IPC181_03440, IPC182_27410, IPC183_07620, IPC184_23205, IPC26_03230, IPC27_25570, IPC29_25820, IPC30_25775, IPC31_26330, IPC32_23895, IPC33_16805, IPC34_07650, IPC35_07645, IPC36_11985, IPC37_11015, IPC38_07925, IPC40_27470, IPC41_03360, IPC42_03360, IPC43_23085, IPC44_22815, IPC45_21905, IPC46_07155, IPC47_03385, IPC48_27355, IPC49_03360, IPC50_30570, IPC51_28835, IPC54_18710, IPC55_30750, IPC56_25845, IPC574_05650, IPC575_13820, IPC576_07870, IPC577_30530, IPC578_05650, IPC579_06085, IPC57_11625, IPC580_13610, IPC582_05645, IPC584_14310, IPC586_05655, IPC589_14875, IPC58_27585, IPC596_07850, IPC597_03535, IPC598_09625, IPC599_08585, IPC59_11860, IPC600_09330, IPC601_08240, IPC602_08660, IPC603_03370, IPC604_03350, IPC605_03350, IPC606_24980, IPC607_31450, IPC608_28595, IPC609_14300, IPC60_11070, IPC610_25045, IPC611_03630, IPC612_03625, IPC613_03625, IPC614_10255, IPC615_03650, IPC616_03650, IPC618_03645, IPC61_27990, IPC620_03665, IPC621_02265, IPC622_09600, IPC623_28715, IPC624_03390, IPC625_10215, IPC627_06380, IPC629_12080, IPC630_04660, IPC632_00200, IPC633_27305, IPC634_00200, IPC64_27165, IPC65_28585, IPC66_27855, IPC67_03650, IPC68_03420, IPC70_17275, IPC71_27005, IPC737_07685, IPC73_27875, IPC74_28395, IPC75_18330, IPC76_17335, IPC77_23945, IPC78_12120, NCTC13621_05232, PA52Ts2_6132, PAMH19_3299
Production host: Escherichia coli (E. coli) / References: UniProt: Q51380, exodeoxyribonuclease III
#3: RNA chain estA mRNA


Mass: 37997.801 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa (bacteria)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of the RNA chaperone Hfq with helper protein Crc assembled on a fragment of estA mRNACOMPLEX#1, #3, #20RECOMBINANT
2RNA-binding protein Hfq and Catabolite repression control proteinCOMPLEX#1-#21RECOMBINANT
3estA mRNACOMPLEX#31RECOMBINANT
Molecular weightValue: 0.26 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pseudomonas aeruginosa (bacteria)287
33Pseudomonas aeruginosa (bacteria)287
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2600 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18rc5_3822: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimera1.13.1model fitting
9cryoSPARC2.15initial Euler assignment
10cryoSPARC2.15final Euler assignment
13REFMACmodel refinement
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46800 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementHighest resolution: 4.5 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00219853
ELECTRON MICROSCOPYf_angle_d0.39627183
ELECTRON MICROSCOPYf_dihedral_angle_d13.9843137
ELECTRON MICROSCOPYf_chiral_restr0.0413068
ELECTRON MICROSCOPYf_plane_restr0.0023284

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