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- PDB-8ra0: Crystal structure of CysF -

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Basic information

Entry
Database: PDB / ID: 8ra0
TitleCrystal structure of CysF
ComponentsAMP-dependent synthetase
KeywordsBIOSYNTHETIC PROTEIN / Beta lactone formation
Function / homology: / ANL, N-terminal domain / AMP-dependent synthetase/ligase / AMP-binding enzyme / AMP-binding enzyme, C-terminal domain superfamily / catalytic activity / CITRATE ANION / AMP-dependent synthetase
Function and homology information
Biological speciesKitasatospora cystarginea (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.89 Å
AuthorsLevy, C.W.
Funding support United Kingdom, European Union, 4items
OrganizationGrant numberCountry
Engineering and Physical Sciences Research CouncilEP/V048929/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/V016083/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/V008552/1 United Kingdom
European CommissionEP/Y023714/1European Union
CitationJournal: Nat.Chem.Biol. / Year: 2024
Title: Cryptic enzymatic assembly of peptides armed with beta-lactone warheads.
Authors: Xu, G. / Torri, D. / Cuesta-Hoyos, S. / Panda, D. / Yates, L.R.L. / Zallot, R. / Bian, K. / Jia, D. / Iorgu, A.I. / Levy, C. / Shepherd, S.A. / Micklefield, J.
History
DepositionNov 30, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Oct 9, 2024Group: Database references / Structure summary
Category: citation / pdbx_entry_details / pdbx_modification_feature
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: AMP-dependent synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,7833
Polymers54,3991
Non-polymers3832
Water4,594255
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area730 Å2
ΔGint6 kcal/mol
Surface area21230 Å2
Unit cell
Length a, b, c (Å)44.052, 68.776, 75.399
Angle α, β, γ (deg.)90.000, 96.833, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein AMP-dependent synthetase


Mass: 54399.336 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Kitasatospora cystarginea (bacteria) / Gene: cysF / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1W6R555
#2: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#3: Chemical ChemComp-FLC / CITRATE ANION


Mass: 189.100 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H5O7
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 255 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
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: 2.09 Å3/Da / Density % sol: 41.05 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop
Details: JCSG Plus A2 0.1 M Sodium citrate 5.5 20 % w/v PEG 3000
Temp details: Cold Room

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.954 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Feb 5, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.954 Å / Relative weight: 1
ReflectionResolution: 1.89→50.65 Å / Num. obs: 35828 / % possible obs: 99.56 % / Redundancy: 6.9 % / Biso Wilson estimate: 26.08 Å2 / CC1/2: 0.992 / Rpim(I) all: 0.0991 / Net I/σ(I): 5.97
Reflection shellResolution: 1.89→1.958 Å / Redundancy: 7 % / Mean I/σ(I) obs: 0.69 / Num. unique obs: 3488 / CC1/2: 0.419 / Rpim(I) all: 0.83 / % possible all: 97.34

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Processing

Software
NameVersionClassification
PHENIX1.21_5207refinement
DIALSdata reduction
DIALSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.89→50.65 Å / SU ML: 0.2642 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 25.9179
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2376 1763 4.93 %
Rwork0.1927 33982 -
obs0.195 35745 99.46 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 33.15 Å2
Refinement stepCycle: LAST / Resolution: 1.89→50.65 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3698 0 26 255 3979
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01193857
X-RAY DIFFRACTIONf_angle_d0.98225256
X-RAY DIFFRACTIONf_chiral_restr0.0598589
X-RAY DIFFRACTIONf_plane_restr0.0112707
X-RAY DIFFRACTIONf_dihedral_angle_d16.47331455
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.89-1.940.3521420.31862470X-RAY DIFFRACTION95.43
1.94-20.32811600.29462599X-RAY DIFFRACTION99.35
2-2.060.30951160.27262580X-RAY DIFFRACTION99.37
2.06-2.140.34361470.25632594X-RAY DIFFRACTION99.75
2.14-2.220.29651270.22892666X-RAY DIFFRACTION99.89
2.22-2.320.26521050.22242614X-RAY DIFFRACTION99.89
2.32-2.440.25581410.21162621X-RAY DIFFRACTION99.82
2.45-2.60.26951320.20782615X-RAY DIFFRACTION99.89
2.6-2.80.25231380.20392623X-RAY DIFFRACTION99.89
2.8-3.080.24841430.1972614X-RAY DIFFRACTION99.96
3.08-3.530.22391320.17212641X-RAY DIFFRACTION99.89
3.53-4.440.17211370.14282654X-RAY DIFFRACTION100
4.44-50.650.19271430.1592691X-RAY DIFFRACTION99.93
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)
13.28604606640.3633143751450.2904222414921.29653613640.2518605924990.7022209300820.004392522603840.0749728432698-0.04174885055630.0629491827716-0.0378022352465-0.005093404986770.09891996281810.05755148735820.04792163305950.2504611452420.00644293365208-0.01228263358210.1563179857610.001403999553650.15497226926836.060583515420.079443557141.3715552106
20.986960448467-0.208251470569-0.05721958463850.818513750873-0.2666265613360.814859516777-0.0219184446518-0.1158101474880.02974708017750.09185259705630.01930415481440.0284999061597-0.0253886096024-0.04236856811490.01499755196560.24002247208-0.00397943205091-0.0176432101590.189837301389-0.01592514216930.17615432554927.705924250934.672796219357.0067761201
30.4075879426190.256877102450.1760034125572.97129532878-0.3301597519420.4442233990620.0867775163965-0.0399539684145-0.02355389296670.041197859068-0.124788803667-0.0148069776828-0.133556341120.0001526038774770.02284290041110.4623133999210.04499309190480.001612976952840.390099832881-0.04285548656150.29174260236142.068259018542.887119774668.2508694027
41.62206026958-0.53521381019-0.9829432411942.221243231070.8772500254783.425091946470.01774320076030.08829174748470.0401027788577-0.0281657355938-0.0531078167357-0.14066587664-0.4383888594760.211840145140.06748498200930.4207299138980.00307628225353-0.008883352722940.2900360500460.01408432695320.25178668467248.578057721656.943197066160.1932833166
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth asym-ID: A / Label asym-ID: A

IDRefine TLS-IDSelection detailsAuth seq-IDLabel seq-ID
11chain 'A' and (resid 1 through 154 )1 - 1541 - 154
22chain 'A' and (resid 155 through 346 )155 - 346155 - 346
33chain 'A' and (resid 347 through 420 )347 - 420347 - 420
44chain 'A' and (resid 421 through 485 )421 - 485421 - 485

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