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- PDB-7mu0: MtbEttA in the ADP bound state -

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Basic information

Entry
Database: PDB / ID: 7mu0
TitleMtbEttA in the ADP bound state
ComponentsEnergy-dependent translational throttle protein EttA
KeywordsHYDROLASE / mycobacterium tuberculosis / ribosome / ABCF ribosome complex / antibiotic
Function / homology
Function and homology information


negative regulation of translational elongation / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / peptidoglycan-based cell wall / ribosome binding / tRNA binding / rRNA binding / translation / ATP hydrolysis activity / ATP binding / plasma membrane / cytosol
Similarity search - Function
Energy-dependent translational throttle protein EttA / ABC-transporter extension domain / ABC transporter / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Energy-dependent translational throttle protein EttA
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.9 Å
AuthorsCui, Z. / Zhang, J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01AI095208 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U24GM116787 United States
CitationJournal: Nat Commun / Year: 2022
Title: Interplay between an ATP-binding cassette F protein and the ribosome from Mycobacterium tuberculosis.
Authors: Zhicheng Cui / Xiaojun Li / Joonyoung Shin / Howard Gamper / Ya-Ming Hou / James C Sacchettini / Junjie Zhang /
Abstract: EttA, energy-dependent translational throttle A, is a ribosomal factor that gates ribosome entry into the translation elongation cycle. A detailed understanding of its mechanism of action is limited ...EttA, energy-dependent translational throttle A, is a ribosomal factor that gates ribosome entry into the translation elongation cycle. A detailed understanding of its mechanism of action is limited due to the lack of high-resolution structures along its ATPase cycle. Here we present the cryo-electron microscopy (cryo-EM) structures of EttA from Mycobacterium tuberculosis (Mtb), referred to as MtbEttA, in complex with the Mtb 70S ribosome initiation complex (70SIC) at the pre-hydrolysis (ADPNP) and transition (ADP-VO) states, and the crystal structure of MtbEttA alone in the post-hydrolysis (ADP) state. We observe that MtbEttA binds the E-site of the Mtb 70SIC, remodeling the P-site tRNA and the ribosomal intersubunit bridge B7a during the ribosomal ratcheting. In return, the rotation of the 30S causes conformational changes in MtbEttA, forcing the two nucleotide-binding sites (NBSs) to alternate to engage each ADPNP in the pre-hydrolysis states, followed by complete engagements of both ADP-VO molecules in the ATP-hydrolysis transition states. In the post-hydrolysis state, the conserved ATP-hydrolysis motifs of MtbEttA dissociate from both ADP molecules, leaving two nucleotide-binding domains (NBDs) in an open conformation. These structures reveal a dynamic interplay between MtbEttA and the Mtb ribosome, providing insights into the mechanism of translational regulation by EttA-like proteins.
History
DepositionMay 14, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 2, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Energy-dependent translational throttle protein EttA
B: Energy-dependent translational throttle protein EttA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,75210
Polymers123,9462
Non-polymers1,8068
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9550 Å2
ΔGint-67 kcal/mol
Surface area50540 Å2
MethodPISA
Unit cell
Length a, b, c (Å)50.060, 106.960, 269.190
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP22121
Space group name HallP22ab(z,x,y)
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y+1/2,-z+1/2
#4: -x,-y+1/2,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and resid 4 through 602)
d_2ens_1(chain "B" and (resid 4 through 551 or resid 601 through 602))

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1PHETHRA2 - 549
d_12ens_1ADPADPB
d_13ens_1ADPADPC
d_21ens_1PHETHRD1 - 548
d_22ens_1ADPADPE
d_23ens_1ADPADPF

NCS oper: (Code: givenMatrix: (-0.999098351545, -0.0420029622425, 0.00618345388994), (-0.0423581585359, 0.996040194888, -0.0781646759939), (-0.00287582068315, -0.0783561186548, -0.996921284919)Vector: ...NCS oper: (Code: given
Matrix: (-0.999098351545, -0.0420029622425, 0.00618345388994), (-0.0423581585359, 0.996040194888, -0.0781646759939), (-0.00287582068315, -0.0783561186548, -0.996921284919)
Vector: 52.4726915104, 6.64220633757, 144.484277489)

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Components

#1: Protein Energy-dependent translational throttle protein EttA / Translational regulatory factor EttA


Mass: 61972.957 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: ettA, Rv2477c / Production host: Escherichia coli (E. coli)
References: UniProt: P9WQK3, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
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: 2.91 Å3/Da / Density % sol: 57.69 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop / Details: 0.1M MES, 0.2M MgCl2, 10% PEG 4000 / PH range: 6.0-6.7

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Data collection

DiffractionMean temperature: 120 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 1.03 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Jul 20, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.03 Å / Relative weight: 1
ReflectionResolution: 2.9→50.06 Å / Num. obs: 33131 / % possible obs: 99.81 % / Redundancy: 2 % / Biso Wilson estimate: 67.88 Å2 / CC1/2: 0.997 / Net I/σ(I): 8.16
Reflection shellResolution: 2.9→3.004 Å / Num. unique obs: 3255 / CC1/2: 0.553

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Processing

Software
NameVersionClassification
PHENIX0.9refinement
REFMAC1.19.1_4122refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4FIN

4fin


Resolution: 2.9→50.06 Å / SU ML: 0.4457 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.1454
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2458 1646 4.97 %
Rwork0.2003 31456 -
obs0.2026 33102 99.84 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 77.45 Å2
Refinement stepCycle: LAST / Resolution: 2.9→50.06 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8573 0 112 0 8685
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00288850
X-RAY DIFFRACTIONf_angle_d0.736612009
X-RAY DIFFRACTIONf_chiral_restr0.04371320
X-RAY DIFFRACTIONf_plane_restr0.00531568
X-RAY DIFFRACTIONf_dihedral_angle_d14.87763319
Refine LS restraints NCSType: Torsion NCS / Rms dev position: 7.87726721023 Å
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.9-2.990.42141560.33992563X-RAY DIFFRACTION99.74
2.99-3.080.33941340.29172561X-RAY DIFFRACTION99.81
3.08-3.190.31881310.26132586X-RAY DIFFRACTION99.74
3.19-3.320.29871330.24652576X-RAY DIFFRACTION99.96
3.32-3.470.26861270.23512587X-RAY DIFFRACTION99.93
3.47-3.650.28041360.22372582X-RAY DIFFRACTION99.89
3.65-3.880.25211100.20272627X-RAY DIFFRACTION99.82
3.88-4.180.241390.1862625X-RAY DIFFRACTION99.93
4.18-4.60.23011590.16542599X-RAY DIFFRACTION100
4.6-5.270.181270.15962641X-RAY DIFFRACTION99.93
5.27-6.630.23441460.19542679X-RAY DIFFRACTION99.89
6.63-50.060.21071480.17562830X-RAY DIFFRACTION99.5
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.88975602074-0.2659737167570.620285509223.035023486610.28779984693.36299083225-0.01698680593050.2322212664910.432632488818-0.00753393959879-0.09638859141030.220081228606-0.1072356510530.2021495077290.1561779722760.330848205-0.011110997471-0.01815358054610.327295766270.02348717759530.31243164123623.450288103580.824894830491.3632059797
20.731195964866-0.618870408828-1.213905637142.003003664812.034712063933.728399462750.153533008927-0.5586951756650.4045689024680.9549248458340.0195238176122-0.0335555781431-0.6370890362350.302467500586-0.04215325570431.11106601703-0.02181963335330.09297852859190.725609769479-0.1095418826350.53767770231925.626112292780.9218917259121.740595573
30.194190560232-0.275002996234-0.32244674581.326936446581.135219872290.838367046883-0.05538876443950.0940129667808-0.03407706525580.106025569824-0.02478254590940.08843342488340.0610202588036-0.0535189446890.09952133515890.371415433318-0.00837221612070.03171561047790.442550490739-0.003916599341660.37650088428323.643301914862.086100244787.6622427326
41.51997190829-0.338612535174-1.002133620941.289218670390.2307065223921.45088644474-0.0591690623132-0.08334991000110.0619594931379-0.150720777006-0.003221519906190.161870195797-0.0201709941959-0.09644217000980.03862053498710.331177491048-0.019568570838-0.08766255215860.35361921978-0.05556183038830.4147256198254.1478005345349.842364907143.0972378909
52.583014553060.1421118015641.3935051072.43313321039-0.3981921266832.0536494304-0.586666324909-0.2206491452721.19448772223-0.367577981349-0.1300357824290.15503743268-1.03120299830.1645831185570.4596451955191.0993646423-0.0151365178328-0.184824819830.454369623036-0.05123706276230.76995449562426.28689286777.30079981345.2668024388
61.91213420403-0.166998673724-0.2049810178613.00918208311-1.342009119914.777414695280.746223807745-0.8267976888140.122638232154-0.284423815543-0.515330594179-0.210806959204-1.253096076090.0454605653675-0.05433078632472.027923788-0.127414837610.1012913516140.9920626267730.1475441235490.7954312433132.115727564582.43426133137.42680851073
72.98256509856-0.3080646064821.025320915712.34949387341-0.1846640957992.61417960283-0.3888282150590.1787479190320.51960504027-0.914022098610.2043754719680.527033195369-0.573036379111-0.0164289282759-0.01191941464510.778645301546-0.0101718037246-0.1682795585790.4126910459220.0623312670070.55849183474621.185361396368.527965451930.7094536624
80.233771689020.347009038849-0.802327548570.794280325498-0.4790810122742.637641816530.0979086387816-0.177460165263-0.2087493587690.259877858437-0.145483959107-0.230024497028-0.2228116037950.3322289313930.01247460955730.408891610155-0.0289645876244-0.1177242957620.5624312173950.0008574731888320.49490311484229.050424588955.755496377262.8140852089
92.157905507590.706899678278-0.7353064766130.7428031177160.0406031289092.01821243141-0.0940385223909-0.214074972733-0.3500108818590.3127765505290.135547724946-0.1655759183320.2216588885530.162997300686-0.02483682915840.4329952670050.117086477636-0.06723602921390.3623174800160.01362282298580.47383838230146.084331297853.752244851896.7689709738
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 3 through 84 )AA3 - 841 - 82
22chain 'A' and (resid 85 through 176 )AA85 - 17683 - 174
33chain 'A' and (resid 177 through 273 )AA177 - 273175 - 271
44chain 'A' and (resid 274 through 549 )AA274 - 549272 - 547
55chain 'B' and (resid 4 through 95 )BD4 - 951 - 92
66chain 'B' and (resid 96 through 130 )BD96 - 13093 - 127
77chain 'B' and (resid 131 through 215 )BD131 - 215128 - 212
88chain 'B' and (resid 216 through 274 )BD216 - 274213 - 271
99chain 'B' and (resid 275 through 549 )BD275 - 549272 - 546

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