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- PDB-8f09: Crystal structure of a trimethoprim-resistant dihydrofolate reduc... -

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Basic information

Entry
Database: PDB / ID: 8f09
TitleCrystal structure of a trimethoprim-resistant dihydrofolate reductase (DHFR) enzyme from an uncultured soil bacterium
ComponentsDihydrofolate reductase
KeywordsOXIDOREDUCTASE / STRUCTURAL GENOMICS / Center for Structural Biology of Infectious Diseases / CSBID / ANTIMICROBIAL RESISTANCE / ANTIBIOTIC RESISTANCE / TRIMETHOPRIM / DHFR / DIHYDROFOLATE REDUCTASE
Function / homology
Function and homology information


dihydrofolate metabolic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / tetrahydrofolate biosynthetic process / NADP binding / cytosol
Similarity search - Function
Dihydrofolate reductase / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase-like domain superfamily
Similarity search - Domain/homology
dihydrofolate reductase
Similarity search - Component
Biological speciesuncultured bacterium (environmental samples)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.45 Å
AuthorsStogios, P.J. / Evdokimova, D. / Borek, D. / Di Leo, R. / Semper, C. / Savchenko, A. / Joachimiak, A. / Satchell, K.J.F. / Center for Structural Biology of Infectious Diseases (CSBID)
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)HHSN272201700060C United States
CitationJournal: To Be Published
Title: Crystal structure of a trimethoprim-resistant dihydrofolate reductase (DHFR) enzyme from an uncultured soil bacterium
Authors: Stogios, P.J.
History
DepositionNov 2, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Other / Structure summary / Category: audit_author / pdbx_SG_project / struct_keywords
Item: _audit_author.name / _pdbx_SG_project.full_name_of_center ..._audit_author.name / _pdbx_SG_project.full_name_of_center / _pdbx_SG_project.initial_of_center / _struct_keywords.text
Revision 1.2Oct 25, 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

Downloads & links

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Assembly

Deposited unit
A: Dihydrofolate reductase
B: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,9327
Polymers38,4522
Non-polymers4805
Water1,29772
1
A: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,5144
Polymers19,2261
Non-polymers2883
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,4183
Polymers19,2261
Non-polymers1922
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)36.073, 41.987, 70.380
Angle α, β, γ (deg.)82.000, 90.000, 72.100
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z

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Components

#1: Protein Dihydrofolate reductase / DHFR


Mass: 19225.906 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) uncultured bacterium (environmental samples)
Plasmid: pMCSG53 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold / References: UniProt: A0A8A1VA22, dihydrofolate reductase
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 72 / Source method: isolated from a natural source / Formula: H2O
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: 2.61 Å3/Da / Density % sol: 52.87 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 20% ammonium sulfate, 0.1 M Bis-Tris pH 8, cryoprotectant ethylene glycol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV / Detector: IMAGE PLATE / Date: Apr 25, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.44→50 Å / Num. obs: 13510 / % possible obs: 93.9 % / Redundancy: 3 % / Biso Wilson estimate: 41.13 Å2 / CC1/2: 0.974 / Rmerge(I) obs: 0.228 / Rpim(I) all: 0.162 / Net I/σ(I): 9.1
Reflection shellResolution: 2.44→2.48 Å / Rmerge(I) obs: 0.924 / Mean I/σ(I) obs: 1.65 / Num. unique obs: 651 / CC1/2: 0.398 / Rpim(I) all: 0.635 / % possible all: 90.7

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Processing

Software
NameVersionClassification
PHENIX1.20_4459refinement
HKL-3000data reduction
HKL-3000data scaling
BALBESphasing
Cootmodel building
PHENIXmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3JWK

3jwk


Resolution: 2.45→34.29 Å / SU ML: 0.4844 / Cross valid method: FREE R-VALUE / σ(F): 2 / Phase error: 42.1455
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.3493 507 4.77 %RANDOM
Rwork0.2889 10131 --
obs0.2918 10638 74.14 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 51.31 Å2
Refinement stepCycle: LAST / Resolution: 2.45→34.29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2710 0 25 72 2807
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00232796
X-RAY DIFFRACTIONf_angle_d0.61063788
X-RAY DIFFRACTIONf_chiral_restr0.0482406
X-RAY DIFFRACTIONf_plane_restr0.0038488
X-RAY DIFFRACTIONf_dihedral_angle_d18.62031026
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.45-2.690.4418810.31161369X-RAY DIFFRACTION40.59
2.69-3.080.37991200.31792361X-RAY DIFFRACTION68.55
3.08-3.880.37211490.28743206X-RAY DIFFRACTION93.85
3.88-34.290.31911570.27913195X-RAY DIFFRACTION93.63
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)
10.448914555037-0.5379135142770.2589452939081.55177870213-1.600026111782.72835845114-0.171711729999-0.05791507513880.2221995994240.694019505186-0.093563121079-0.0712800210162-0.349346902096-0.2716095997770.1173004203020.0210041192188-0.0717759095157-0.009770040180170.199633543122-0.1706599876210.322651775176-4.07595455297-6.43714311251-11.951605944
25.712288258540.786856876076-2.03063267526.293998822731.59206130885.223573975190.4907157706010.09135103857850.943036282564-0.182484251533-0.0466166344459-0.616294744121-0.3537292636290.15607363322-0.4288737279580.174077696872-0.0886900898786-0.06650310568860.5701097128940.008156694877940.3675705956565.77563460633-0.496191869135-16.3429040093
31.65398323745-0.667762434487-1.639329017033.982606318082.715713578262.768626886-0.0177734359994-0.1789166176510.3235294666650.462794954170.334191992818-0.4920333606050.570459280611.021456031440.1933481544630.1038761453140.0606313569506-0.1726010370210.574755006701-0.003600642203030.29061096519510.574699338-1.28003964681-12.431938204
44.638835792681.290095032534.075497089791.93602490002-0.9673966449196.38496960591-0.0734027436578-0.751103047870.83495979737-0.239478009893-0.6899068492690.0550715108551-0.5024997916020.919315467370.09517587178150.370740470458-0.0397823765007-0.07026599801160.79869883662-0.1323078479890.225631004419.6923040734111.1368661344-19.1984752905
55.28613355586-0.495342653538-0.7689903135677.66142958933-0.8153246796824.26551804112-0.000235211900579-0.08123928383380.639826882945-0.2473424652910.0281430169656-0.6829661551230.03131992305840.2182667447830.01630545980910.1872995405010.0080769139884-0.009064334755120.591610410854-0.04643700951810.1925581732496.13067224109-1.77217727572-26.5664533651
62.04106227855-1.98153925316-2.549805124223.473207156632.156736401423.452492699980.275391719351-0.01931484467180.127617909973-0.557271003202-0.031525114011-0.472830272701-0.316767070467-0.72119755487-0.2565112003860.105245004668-0.1348971399350.009993114597960.529584781086-0.2124099496540.31929033794-9.45303394112-3.52843627267-17.6243840739
70.8506229747620.0789228300875-1.623359925892.499550305231.26748692733.96671840365-0.08527890508370.368655878184-0.272306961068-0.05897650662050.480411940890.008720601524380.478137430599-0.571621022837-0.2482149320020.098242748822-0.01552110775430.07298911214750.4436175074180.08868453897320.342603160888-10.5877383765-13.713370086-14.8993990126
80.6030896768970.373731197339-0.2280854108621.10682856626-0.4954292838112.265814725650.0552122055401-0.2577830200830.1275763668540.315073576645-0.0512765757330.1318067550620.9649345091220.1347249086450.09043155975430.238467447128-0.02289105999970.2751885840230.606255074122-0.04442390691590.08342385318176.678839861695.0301803401621.335022684
91.627756962851.899893238380.2287611008522.88722136287-0.7932904656641.814761717770.333817503493-0.1210885380540.302241440440.526718215953-0.8995761227570.649181508139-0.370036718656-0.0721466547357-0.01072329978820.313033290480.1604431241340.02957227559780.320983152648-0.119873001340.274625204992-6.619415640454.7785773198421.1378072313
106.10895292976-0.6991715353340.8539601999813.808509186441.909759251091.235405928450.0238289095826-0.277983827473-0.816669722636-0.03982937458670.0785267990910.1094631739230.2945481011890.215148181218-0.1001682673360.208071893160.03078618429830.01918420452730.5777748106830.05204583272230.12457954661-7.33707554374-4.2710914401112.573399657
112.193409979490.4774663206490.505909292821.45285185159-0.8840551827644.93909705760.266470842650.3272009013830.391408027424-0.141683304444-0.08334394813070.08088545281530.1516354507841.15961083206-0.1359463058510.1809677906670.0865678333818-0.004316970700140.455914008766-0.02462909737140.23742401539610.87798458516.3792569108114.6495624641
120.957809258290.08517850168160.1718291020581.82391577707-0.2802948646352.313205538290.09765906629050.0630553809267-0.3910261376830.18993772172-0.166824751909-0.02520233803140.3540192465410.314302570682-0.1280184953430.2876383510810.0239727621717-0.1434350359460.1188788052120.1302668565160.3640860983078.7508281772315.465355672421.7169145295
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 0 through 35 )AA0 - 351 - 36
22chain 'A' and (resid 36 through 49 )AA36 - 4937 - 50
33chain 'A' and (resid 50 through 66 )AA50 - 6651 - 67
44chain 'A' and (resid 67 through 77 )AA67 - 7768 - 78
55chain 'A' and (resid 78 through 103 )AA78 - 10379 - 104
66chain 'A' and (resid 104 through 132 )AA104 - 132105 - 133
77chain 'A' and (resid 133 through 166 )AA133 - 166134 - 167
88chain 'B' and (resid 0 through 25 )BB0 - 251 - 26
99chain 'B' and (resid 26 through 66 )BB26 - 6627 - 67
1010chain 'B' and (resid 67 through 103 )BB67 - 10368 - 104
1111chain 'B' and (resid 104 through 148 )BB104 - 148105 - 149
1212chain 'B' and (resid 149 through 166 )BB149 - 166150 - 167

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