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- PDB-8ry5: CryoEM structure of M. smegmatis GMP reductase in complex with GM... -

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Basic information

Entry
Database: PDB / ID: 8ry5
TitleCryoEM structure of M. smegmatis GMP reductase in complex with GMP and ATP at pH 6.6, compressed conformation.
ComponentsGMP reductase
KeywordsOXIDOREDUCTASE / GMP reductase / GuaB1 / CBS domain / Mycobacterium smegmatis
Function / homology
Function and homology information


GMP reductase / GMP reductase activity / IMP salvage / IMP dehydrogenase activity / purine ribonucleoside salvage / cytosol
Similarity search - Function
GMP reductase-like / : / Inosine-5'-monophosphate dehydrogenase / IMP dehydrogenase/GMP reductase / IMP dehydrogenase / GMP reductase domain / IMP dehydrogenase / GMP reductase domain / CBS domain superfamily / CBS domain / CBS domain / CBS domain profile. / Aldolase-type TIM barrel
Similarity search - Domain/homology
GUANOSINE-5'-MONOPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / GMP reductase
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.48 Å
AuthorsDolezal, M. / Kouba, T. / Pichova, I.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Union (EU)LX22NPO5103European Union
CitationJournal: Nat Commun / Year: 2026
Title: Structural basis for allosteric regulation of mycobacterial guanosine 5´-monophosphate reductase by ATP and GTP.
Authors: Michal Doležal / Zdeněk Knejzlík / Tomáš Kouba / Anatolij Filimoněnko / Hana Šváchová / Matteo Dedola / Martin Klíma / Iva Pichová /
Abstract: Guanosine 5'-monophosphate reductase (GMPR) is a crucial enzyme in the purine salvage pathway that catalyses the NADPH-dependent conversion of GMP to IMP, thereby contributing to purine nucleotide ...Guanosine 5'-monophosphate reductase (GMPR) is a crucial enzyme in the purine salvage pathway that catalyses the NADPH-dependent conversion of GMP to IMP, thereby contributing to purine nucleotide homeostasis. Mycobacterium smegmatis GMPR (MsmGMPR) contains a regulatory cystathionine β-synthase (CBS) domain, which mediates allosteric modulation by ATP and GTP. However, MsmGMPR exhibits an atypical tertiary structure that is incompatible with the acknowledged regulatory mechanisms of IMPDH/GMPR family enzymes. Here, we combine X-ray crystallography, cryogenic electron microscopy, and biochemical binding assays to elucidate the molecular basis of MsmGMPR regulation by ATP and GTP. We show that ATP stabilises a compressed conformation that inhibits the enzyme by restricting access to the active site and preventing NADPH binding. In contrast, GTP counteracts ATP binding, promoting an active conformation that enables catalysis. Our results provide insight into how MsmGMPR senses and responds to the purine nucleotide balance, revealing a distinct utilisation of the CBS domain compared with its typical role in IMPDH/GMPR enzymes.
History
DepositionFeb 8, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 20, 2025Provider: repository / Type: Initial release
Revision 1.0Aug 20, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 20, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Aug 20, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 20, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 20, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 20, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 20, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
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Revision 1.1Apr 22, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Apr 22, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GMP reductase
B: GMP reductase
C: GMP reductase
D: GMP reductase
E: GMP reductase
F: GMP reductase
G: GMP reductase
H: GMP reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)421,22324
Polymers414,2598
Non-polymers6,96316
Water3,855214
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails (eV)
d_1ens_1chain C
d_2ens_1chain A
d_1ens_2chain B
d_2ens_2chain D
d_1ens_3chain G
d_2ens_3chain E
d_1ens_4chain F
d_2ens_4chain H

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11ens_1VALVALALAALACC2 - 4812 - 481
d_12ens_15GP5GP5GP5GPCM501
d_13ens_1ATPATPATPATPCN502
d_21ens_1VALVALALAALAAA2 - 4812 - 481
d_22ens_15GP5GP5GP5GPAI501
d_23ens_1ATPATPATPATPAJ502
d_11ens_2VALVALALAALABB2 - 4812 - 481
d_12ens_25GP5GP5GP5GPBK501
d_13ens_2ATPATPATPATPBL502
d_21ens_2VALVALALAALADD2 - 4812 - 481
d_22ens_25GP5GP5GP5GPDO501
d_23ens_2ATPATPATPATPDP502
d_11ens_3VALVALALAALAGG2 - 4812 - 481
d_12ens_35GP5GP5GP5GPGU501
d_13ens_3ATPATPATPATPGV502
d_21ens_3VALVALALAALAEE2 - 4812 - 481
d_22ens_35GP5GP5GP5GPEQ501
d_23ens_3ATPATPATPATPER502
d_11ens_4VALVALALAALAFF2 - 4812 - 481
d_12ens_45GP5GP5GP5GPFS501
d_13ens_4ATPATPATPATPFT502
d_21ens_4VALVALALAALAHH2 - 4812 - 481
d_22ens_45GP5GP5GP5GPHW501
d_23ens_4ATPATPATPATPHX502

NCS ensembles :
ID
ens_1
ens_2
ens_3
ens_4

NCS oper:
IDCodeMatrixVector
1given(-0.999999072018, 0.000315172795719, 0.00132537885071), (-0.000315451194104, -0.999999928227, -0.000209848129806), (0.00132531261716, -0.000210266027412, 0.999999099667)366.522061377, 366.856727352, -0.169087877179
2given(-0.999999540918, -0.000121693697247, -0.000950449875392), (0.000122083485232, -0.999999908473, -0.000410061684377), (-0.000950399886477, -0.000410177530358, 0.999999464247)366.946658829, 366.821145093, 0.260492839025
3given(-0.99999974872, -0.000350946340503, -0.000615952101443), (0.000351371048884, -0.999999700535, -0.000689542553066), (-0.000615709924551, -0.000689758807534, 0.999999572567)366.983386319, 366.864984586, 0.24299579012
4given(-0.999999890708, 0.000439549440756, 0.000159314379808), (-0.000439581164168, -0.999999883555, -0.0001991443111), (0.000159226827486, -0.000199214320935, 0.99999996748)366.671265014, 366.904577021, 0.0107463425065

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Components

#1: Protein
GMP reductase / Guanosine 5'-monophosphate reductase / GMPR


Mass: 51782.434 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Details: Guanosine 5'-monophosphate reductase / Source: (gene. exp.) Mycolicibacterium smegmatis (bacteria) / Strain: ATCC 700084 / mc(2)155 / Gene: guaB1, MSMEG_3634, MSMEI_3548 / Plasmid: pTriex / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0QYE8, GMP reductase
#2: Chemical
ChemComp-5GP / GUANOSINE-5'-MONOPHOSPHATE


Mass: 363.221 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H14N5O8P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 214 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Msm GMPR with GMP and ATP at pH 6.6 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria) / Strain: ATCC 700084 / mc(2)155
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pTriex
Buffer solutionpH: 6.6
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acidHEPES1
2100 mMPotassium chlorideKCl1
32 mMMagnesium chlorideMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 20 mg/ml Msm GMPR with 2 mM GMP and 5 mM ATP in the storage buffer (50 mM Tris, pH 8.0, 2.5 mM TCEP) was diluted with the cryoEM buffer (50 mM HEPES, pH 6.6, 100 mM KCl, 2 mM MgCl2).
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 30 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
7MOLREP11.9.02model fitting
8UCSF ChimeraX1.5model fitting
11RELION4final Euler assignment
14PHENIX1.21rc1_4985model refinement
15Coot0.9.8.1 ELmodel refinement
16ISOLDE1.5model refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107511 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Details: The initial structure was obtained by fitting an appropriate model into the cryo-EM map using MolRep and ChimeraX. The structure was then refined by iterative manual rebuilding in Coot and ...Details: The initial structure was obtained by fitting an appropriate model into the cryo-EM map using MolRep and ChimeraX. The structure was then refined by iterative manual rebuilding in Coot and Isolde, and automatic refinement in phenix.real_space_refine.
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 38.57 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001428840
ELECTRON MICROSCOPYf_angle_d0.412539424
ELECTRON MICROSCOPYf_chiral_restr0.04014632
ELECTRON MICROSCOPYf_plane_restr0.00345208
ELECTRON MICROSCOPYf_dihedral_angle_d12.56810488
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2CCELECTRON MICROSCOPYNCS constraints2.40376923566E-12
ens_2d_2BBELECTRON MICROSCOPYNCS constraints1.47171855315E-10
ens_3d_2GGELECTRON MICROSCOPYNCS constraints1.05534989812E-12
ens_4d_2FFELECTRON MICROSCOPYNCS constraints2.4625340412E-12

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