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- PDB-8db3: Crystal structure of KaiC with truncated C-terminal coiled-coil domain -

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Basic information

Entry
Database: PDB / ID: 8db3
TitleCrystal structure of KaiC with truncated C-terminal coiled-coil domain
Components(Circadian clock protein KaiC) x 2
KeywordsCIRCADIAN CLOCK PROTEIN / autokinase
Function / homology
Function and homology information


kinase activity / non-specific serine/threonine protein kinase / ATP hydrolysis activity / ATP binding / metal ion binding
Similarity search - Function
Circadian clock protein kinase KaiC / : / : / KaiC domain / KaiC domain profile. / KaiC-like domain / KaiC / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain ...Circadian clock protein kinase KaiC / : / : / KaiC domain / KaiC domain profile. / KaiC-like domain / KaiC / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / non-specific serine/threonine protein kinase
Similarity search - Component
Biological speciesCereibacter sphaeroides (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.9 Å
AuthorsPadua, R.A.P. / Grant, T. / Pitsawong, W. / Hoemberger, M.S. / Otten, R. / Bradshaw, N. / Grigorieff, N. / Kern, D.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2023
Title: From primordial clocks to circadian oscillators.
Authors: Warintra Pitsawong / Ricardo A P Pádua / Timothy Grant / Marc Hoemberger / Renee Otten / Niels Bradshaw / Nikolaus Grigorieff / Dorothee Kern /
Abstract: Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary ...Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and a central component of the clock. Subsequent additions of KaiB and KaiA regulate the phosphorylation state of KaiC for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that only possess KaiBC. However, there are reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here we investigate the primordial circadian clock in Rhodobacter sphaeroides, which contains only KaiBC, to elucidate its inner workings despite missing KaiA. Using a combination of X-ray crystallography and cryogenic electron microscopy, we find a new dodecameric fold for KaiC, in which two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the carboxy-terminal extension of KaiC and serves as an ancient regulatory moiety that is later superseded by KaiA. A coiled-coil register shift between daytime and night-time conformations is connected to phosphorylation sites through a long-range allosteric network that spans over 140 Å. Our kinetic data identify the difference in the ATP-to-ADP ratio between day and night as the environmental cue that drives the clock. They also unravel mechanistic details that shed light on the evolution of self-sustained oscillators.
History
DepositionJun 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 19, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 25, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation_author / pdbx_initial_refinement_model
Item: _citation_author.identifier_ORCID
Revision 1.4Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Circadian clock protein KaiC
B: Circadian clock protein KaiC
C: Circadian clock protein KaiC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,7049
Polymers161,1413
Non-polymers2,5636
Water00
1
A: Circadian clock protein KaiC
B: Circadian clock protein KaiC
C: Circadian clock protein KaiC
hetero molecules

A: Circadian clock protein KaiC
B: Circadian clock protein KaiC
C: Circadian clock protein KaiC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)327,40918
Polymers322,2836
Non-polymers5,12612
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_555-x,y,-z+1/21
Buried area42400 Å2
ΔGint-131 kcal/mol
Surface area83430 Å2
MethodPISA
Unit cell
Length a, b, c (Å)94.060, 197.280, 150.190
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2

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Components

#1: Protein Circadian clock protein KaiC


Mass: 53687.105 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cereibacter sphaeroides (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: B9KWX8
#2: Protein Circadian clock protein KaiC


Mass: 53767.082 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cereibacter sphaeroides (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: B9KWX8
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationY

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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: 2.16 Å3/Da / Density % sol: 43.11 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop
Details: 0.2 M Ammonium acetate, 0.1 M Sodium citrate tribasic dihydrate pH 5.6, 30% w/v Polyethylene glycol 4,000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.1 / Wavelength: 0.8855 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Nov 3, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8855 Å / Relative weight: 1
ReflectionResolution: 2.9→59.75 Å / Num. obs: 31367 / % possible obs: 99.89 % / Redundancy: 7.2 % / Biso Wilson estimate: 49.59 Å2 / CC1/2: 0.976 / Net I/σ(I): 5.54
Reflection shellResolution: 2.9→3.004 Å / Mean I/σ(I) obs: 1.15 / Num. unique obs: 3103 / CC1/2: 0.343

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
MOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1tf7

1tf7


Resolution: 2.9→59.75 Å / SU ML: 0.4098 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 30.6042
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2946 1999 6.38 %
Rwork0.2359 29344 -
obs0.2397 31343 99.9 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 52.43 Å2
Refinement stepCycle: LAST / Resolution: 2.9→59.75 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10131 0 162 0 10293
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.004810475
X-RAY DIFFRACTIONf_angle_d0.77314172
X-RAY DIFFRACTIONf_chiral_restr0.04411610
X-RAY DIFFRACTIONf_plane_restr0.00521813
X-RAY DIFFRACTIONf_dihedral_angle_d9.58721497
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.9-2.970.39271400.34992062X-RAY DIFFRACTION100
2.97-3.050.3881430.33342088X-RAY DIFFRACTION99.96
3.05-3.140.38341400.30512065X-RAY DIFFRACTION99.95
3.14-3.240.33791410.28942062X-RAY DIFFRACTION99.91
3.24-3.360.36931420.28622093X-RAY DIFFRACTION99.96
3.36-3.490.31851410.25662068X-RAY DIFFRACTION99.95
3.49-3.650.33111430.24032088X-RAY DIFFRACTION100
3.65-3.850.27961400.23412065X-RAY DIFFRACTION99.95
3.85-4.090.24261420.20952084X-RAY DIFFRACTION99.96
4.09-4.40.24641430.1952100X-RAY DIFFRACTION100
4.4-4.850.23451440.18272100X-RAY DIFFRACTION100
4.85-5.550.29671440.20342130X-RAY DIFFRACTION100
5.55-6.980.30831440.23642114X-RAY DIFFRACTION99.96
6.99-59.750.25861520.21912225X-RAY DIFFRACTION99.12
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)
11.71963412674-0.652230695540.05370249648112.059952881290.01878900550332.655728477890.1213478627660.280117257313-0.262887865983-0.0559931280859-0.0674809084976-0.1162224237990.2463744352840.4272357147470.008569935749940.1679330808920.06286384325930.01309909132870.452897111869-0.07378852213830.23453997530528.340496257718.934407041329.2446082147
20.6647919672471.03966728002-0.1066196479152.13125611576-0.1167144076870.406636856669-0.1670928568840.240408622869-0.0194437580918-0.1773898781890.392192784716-0.3315030358010.014185132370.5885749830060.2594404694590.231492746996-0.133082375250.0543712534051.237709155070.1498198749750.29731842772132.35723497660.163233540127.8939195648
32.370652868230.7794062629011.152806274552.267195400971.265716846765.47124983221-0.01678050839560.323806214495-0.238079093762-0.1201412309720.1544095791150.04554673233840.242464743220.549453178132-0.0801972093250.2616029866330.0284882156681-0.006510645910590.7689910029050.1667862375510.29842624657826.53507779163.585565299921.8252355653
41.58750250655-0.210583366179-0.2101295916552.282096555630.4897031926592.03795727598-0.006761620114520.250484057093-0.156216180406-0.0309471542431-0.01481223491710.06146118876190.2175889818030.1194150972440.002095143958170.181377987379-0.02542854155650.01497800430790.331769723544-0.03077120342570.1811666549657.291131745119.71096070988.49426521083
50.9633766217262.32615978765-0.4707149322976.56489726871-1.484093870770.352214603053-0.2980261219230.360318537792-0.132555502075-0.6422208895120.176950851269-0.564310470391-0.02112084318720.2844094979160.1379603233780.261956271238-0.01722079843940.01309542667030.6900710745180.06482999838190.328491745259.4957376009657.6715303543.7983579697
65.21532815687-0.156395824626-0.601705370864.78877239122-0.0929886139163.17106655848-0.24650561038-0.0309755414926-0.3801802422360.111813734189-0.000585585810175-0.1032199227670.1843027061820.1289029587670.2352397661010.16296198591-0.03250728420350.04467451563050.4845561126330.08133822912930.219105098801-0.38670868667462.98237676198.27688204572
72.117981584180.2300618122370.5969060695091.89321651040.446682871142.230746809930.09204665896940.135378693551-0.251003443681-0.128857342222-0.06364099742240.1934824860260.121409356114-0.327799552349-0.05606707165450.195348842181-0.0358802647212-0.00489076706760.3583387615480.01212096563450.177633646055-21.289166538118.693337581917.2805155126
80.111232341710.298153996880.1177680600085.72364245843-1.363893811410.603953814367-0.0574972309930.199899979252-0.0140933867882-0.8699012362740.295777850005-0.09871079961010.1352667580130.127787198878-0.09375455517720.2958807962990.05463215931380.02939281715560.5716137757450.04151259373160.367804002529-24.729272097957.461578032712.3334625726
93.239866484180.414276664121-0.626679717352.40495215486-0.7721286779775.09350265977-0.0867700560992-0.215949483433-0.0363414904380.108866003027-0.00629485414559-0.02349930098290.705293425134-0.05335434917330.02564220730410.287908650202-0.005637325028210.0081046908410.487128207813-0.05763299101070.190423194426-26.411180373463.02097728323.048593368
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 2 through 219 )AA2 - 2191 - 209
22chain 'A' and (resid 220 through 277 )AA220 - 277210 - 264
33chain 'A' and (resid 278 through 463 )AA278 - 463265 - 434
44chain 'B' and (resid 1 through 219 )BD1 - 2191 - 205
55chain 'B' and (resid 220 through 266 )BD220 - 266206 - 252
66chain 'B' and (resid 267 through 463 )BD267 - 463253 - 439
77chain 'C' and (resid 2 through 219 )CG2 - 2191 - 213
88chain 'C' and (resid 220 through 266 )CG220 - 266214 - 260
99chain 'C' and (resid 267 through 463 )CG267 - 463261 - 446

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