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- PDB-8c51: Trypanosoma brucei IMP dehydrogenase (cyto) crystallized in High ... -

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Basic information

Entry
Database: PDB / ID: 8c51
TitleTrypanosoma brucei IMP dehydrogenase (cyto) crystallized in High Five cells revealing native ligands ATP, GDP and phosphate. Diffraction data collection at 100 K in cellulo
ComponentsInosine-5'-monophosphate dehydrogenase
KeywordsTRANSFERASE / IMP dehydrogenase / purine biosynthetic enzyme / native cofactors
Function / homology
Function and homology information


IMP dehydrogenase activity / IMP dehydrogenase / glycosome / GMP biosynthetic process / nucleotide binding / metal ion binding
Similarity search - Function
IMP dehydrogenase / GMP reductase domain / Inosine-5'-monophosphate dehydrogenase / IMP dehydrogenase / GMP reductase, conserved site / IMP dehydrogenase / GMP reductase signature. / IMP dehydrogenase/GMP reductase / IMP dehydrogenase / GMP reductase domain / Domain in cystathionine beta-synthase and other proteins. / CBS domain / CBS domain / CBS domain profile. / Aldolase-type TIM barrel
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / GUANOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Inosine-5'-monophosphate dehydrogenase
Similarity search - Component
Biological speciesTrypanosoma brucei (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 Å
AuthorsLahey-Rudolph, J.M. / Schoenherr, R. / Boger, J. / Harms, M. / Kaiser, J. / Nachtschatt, S. / Wobbe, M. / Duden, R. / Koenig, P. / Bourenkov, G. ...Lahey-Rudolph, J.M. / Schoenherr, R. / Boger, J. / Harms, M. / Kaiser, J. / Nachtschatt, S. / Wobbe, M. / Duden, R. / Koenig, P. / Bourenkov, G. / Schneider, T. / Redecke, L.
Funding support Germany, 3items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research05K18FLA, InCellSX Germany
Joachim Herz StiftungPhD scholarship Germany
German Research Foundation (DFG)EXC306 Germany
CitationJournal: Nat Commun / Year: 2024
Title: A streamlined approach to structure elucidation using in cellulo crystallized recombinant proteins, InCellCryst.
Authors: Schonherr, R. / Boger, J. / Lahey-Rudolph, J.M. / Harms, M. / Kaiser, J. / Nachtschatt, S. / Wobbe, M. / Duden, R. / Konig, P. / Bourenkov, G. / Schneider, T.R. / Redecke, L.
History
DepositionJan 6, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Inosine-5'-monophosphate dehydrogenase
B: Inosine-5'-monophosphate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,2198
Polymers111,1292
Non-polymers2,0916
Water1,13563
1
A: Inosine-5'-monophosphate dehydrogenase
B: Inosine-5'-monophosphate dehydrogenase
hetero molecules

A: Inosine-5'-monophosphate dehydrogenase
B: Inosine-5'-monophosphate dehydrogenase
hetero molecules

A: Inosine-5'-monophosphate dehydrogenase
B: Inosine-5'-monophosphate dehydrogenase
hetero molecules

A: Inosine-5'-monophosphate dehydrogenase
B: Inosine-5'-monophosphate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)452,87832
Polymers444,5158
Non-polymers8,36324
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_565-x,-y+1,z1
crystal symmetry operation3_555-y+1/2,x+1/2,z1
crystal symmetry operation4_455y-1/2,-x+1/2,z1
Buried area45530 Å2
ΔGint-240 kcal/mol
Surface area131240 Å2
MethodPISA
Unit cell
Length a, b, c (Å)205.750, 205.750, 92.100
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number90
Space group name H-MP4212
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z
#3: y+1/2,-x+1/2,z
#4: x+1/2,-y+1/2,-z
#5: -x+1/2,y+1/2,-z
#6: -x,-y,z
#7: y,x,-z
#8: -y,-x,-z
Components on special symmetry positions
IDModelComponents
11B-729-

HOH

Noncrystallographic symmetry (NCS)NCS oper: (Code: givenMatrix: (0.499849396732, -0.866084647687, -0.00692557782897), (-0.866089505772, -0.49987830009, 0.00326390723311), (-0.00628876601835, 0.00436670821762, -0.999970691211)Vector: ...NCS oper: (Code: given
Matrix: (0.499849396732, -0.866084647687, -0.00692557782897), (-0.866089505772, -0.49987830009, 0.00326390723311), (-0.00628876601835, 0.00436670821762, -0.999970691211)
Vector: 89.0664859815, 154.470840524, 12.4839386356)

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Components

#1: Protein Inosine-5'-monophosphate dehydrogenase / IMP dehydrogenase / IMPD / IMPDH


Mass: 55564.375 Da / Num. of mol.: 2
Mutation: T488S; C-terminal A expression tag insertion, and deletion of C-terminal SKL, N-terminal Gly expression tag insertion,
Source method: isolated from a genetically manipulated source
Details: sequence lacking SKL transport signal for targeting the peroxysome, thus cytoplasmic construct. Cloning-derived G and A artefacts at N- and C-termini
Source: (gene. exp.) Trypanosoma brucei (eukaryote) / Tissue: Glycosome / Plasmid: IMPDH cyto / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): BTI-Tn-5B1-4 / References: UniProt: P50098, IMP dehydrogenase
#2: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 63 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 4.5 Å3/Da / Density % sol: 72 % / Description: thick needles with a square base
Crystal growTemperature: 300 K / Method: in cell
Details: in cellulo rBV IMPDH cyto (MOI 1) infected High Five cells, adhesion culture.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: Y
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.976 Å
DetectorType: DECTRIS EIGER2 S 16M / Detector: PIXEL / Date: Nov 26, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 2.399→77.82 Å / Num. obs: 77541 / % possible obs: 99.88 % / Redundancy: 1797 % / CC1/2: 0.9973 / CC star: 0.9993 / R split: 0.0862 / Net I/σ(I): 10.06
Reflection shellResolution: 2.399→2.456 Å / Redundancy: 1018 % / Mean I/σ(I) obs: 1.809 / Num. unique obs: 5119 / CC1/2: 0.2331 / CC star: 0.6149 / R split: 0.089 / % possible all: 99.32
Serial crystallography sample deliveryDescription: micro-mesh / Method: fixed target
Serial crystallography sample delivery fixed targetDetails: semi-automated cryo-cooling procedure / Motion control: MD3-diffractometer
Sample dehydration prevention: 90 % humidity air stream during blotting
Sample holding: mesh / Sample solvent: ESF921 cell culture medium / Sample unit size: 25 µm / Support base: goniometer
Serial crystallography data reductionFrames indexed: 75741 / Frames total: 96949 / Lattices indexed: 82831

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
MxCuBEdata collection
CrystFELCrystFEL 0.10.1data reduction
CrystFELCrystFEL 0.9.1data scaling
Coot0.9.7model building
PDB_EXTRACTdata extraction
PHENIX1.19.2_4158phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1JCN

1jcn


Resolution: 2.4→72.74 Å / SU ML: 0.43 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 30.01 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2404 1120 1.44 %1
Rwork0.2252 ---
obs0.2254 77524 99.92 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.4→72.74 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6737 0 128 63 6928
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0037035
X-RAY DIFFRACTIONf_angle_d0.6249549
X-RAY DIFFRACTIONf_dihedral_angle_d7.979991
X-RAY DIFFRACTIONf_chiral_restr0.0411099
X-RAY DIFFRACTIONf_plane_restr0.0041209
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.4-2.510.42531360.39279381X-RAY DIFFRACTION99
2.51-2.640.34361380.35879414X-RAY DIFFRACTION100
2.64-2.810.37981390.33459435X-RAY DIFFRACTION100
2.81-3.020.31051390.30119478X-RAY DIFFRACTION100
3.02-3.330.33631400.27089507X-RAY DIFFRACTION100
3.33-3.810.23781400.2219554X-RAY DIFFRACTION100
3.81-4.80.17711420.18259642X-RAY DIFFRACTION100
4.8-72.740.19421460.18099993X-RAY DIFFRACTION100
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)
11.86190.60730.57191.37540.22531.15370.001-0.1828-0.02090.0576-0.0159-0.04280.05330.000600.6730.0660.06550.62920.05790.631424.564289.3944-19.8474
20.8617-0.68651.111.0237-0.57550.69840.0238-0.1780.24470.0879-0.0219-0.44820.10490.1628-0.00010.90960.0935-0.0160.83820.04880.847653.551992.0169-0.406
31.75240.25980.65672.7492-0.05231.5975-0.01490.18670.1107-0.1076-0.2825-0.20370.06010.1938-0.00020.82970.14610.0150.93370.11510.82960.500279.52710.1781
42.41071.11710.19291.2372-0.61011.0249-0.05630.06350.11420.25390.0139-0.37450.00940.3255-0.00030.66150.0485-0.03720.72410.03360.728641.6714102.6847-19.2778
51.47050.15550.97742.03220.05680.8875-0.17660.02050.04490.0123-0.0259-0.19120.1140.489-0.00010.57050.0338-0.00660.66690.02520.640925.5354102.6017-25.0042
62.03140.69341.27570.71790.14650.93040.1172-0.3322-0.03270.3216-0.10590.07090.2172-0.1996-0.00010.72520.0440.03260.71110.06430.679121.667989.3799-12.3996
71.0116-0.0375-0.14222.31090.70461.01680.12610.1343-0.1415-0.1351-0.1105-0.11420.0443-0.0510.00190.63610.03110.05260.66350.03620.647124.105488.501532.5792
82.3427-0.4238-0.87210.54860.04451.2031-0.0921-0.0161-0.03960.0672-0.040.07680.28740.0458-0.00060.93550.12870.08590.84180.01460.75246.139162.065312.7085
90.4884-0.31570.3491.42820.26521.34710.13090.1152-0.4884-0.236-0.106-0.24490.2991-0.0582-0.00020.84410.02660.05430.7426-0.05240.773526.885966.643227.5547
100.9844-1.2442-0.52482.94380.76631.6421-0.1042-0.0171-0.1544-0.0010.1140.02190.2133-0.02510.00010.70020.00590.04550.58910.00930.696514.168673.698537.194
111.3801-0.2681-0.14860.84860.69010.5483-0.21530.1952-0.0591-0.24520.0447-0.12220.3031-0.270.00020.650.06570.08070.68920.03330.686619.584586.227330.2523
120.36840.03610.08791.71770.95631.5272-0.16750.1818-0.0389-0.41960.1828-0.0067-0.11220.4478-0.00040.78420.02740.05270.73640.05160.653323.183792.033224.5967
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 3 through 92 )
2X-RAY DIFFRACTION2chain 'A' and (resid 93 through 132 )
3X-RAY DIFFRACTION3chain 'A' and (resid 133 through 226 )
4X-RAY DIFFRACTION4chain 'A' and (resid 227 through 300 )
5X-RAY DIFFRACTION5chain 'A' and (resid 301 through 363 )
6X-RAY DIFFRACTION6chain 'A' and (resid 364 through 493 )
7X-RAY DIFFRACTION7chain 'B' and (resid 3 through 92 )
8X-RAY DIFFRACTION8chain 'B' and (resid 93 through 226 )
9X-RAY DIFFRACTION9chain 'B' and (resid 227 through 260 )
10X-RAY DIFFRACTION10chain 'B' and (resid 261 through 350 )
11X-RAY DIFFRACTION11chain 'B' and (resid 351 through 385 )
12X-RAY DIFFRACTION12chain 'B' and (resid 386 through 493 )

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