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- EMDB-46711: Class Ia ribonucleotide reductase with mechanism-based inhibitor N3CDP -

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Basic information

Entry
Database: EMDB / ID: EMD-46711
TitleClass Ia ribonucleotide reductase with mechanism-based inhibitor N3CDP
Map dataFinal map following density modification
Sample
  • Complex: Active state of class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP
    • Protein or peptide: Ribonucleoside-diphosphate reductase 1 subunit alpha
    • Protein or peptide: Ribonucleoside-diphosphate reductase 1 subunit beta
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: UNKNOWN LIGAND
  • Ligand: 4-amino-1-{(3xi)-3-C-amino-2-deoxy-5-O-[(S)-hydroxy(phosphonooxy)phosphoryl]-beta-D-threo-pentofuranosyl}pyrimidin-2(1H)-one
  • Ligand: MU-OXO-DIIRON
  • Ligand: water
Keywordsribonucleotide reductase / class Ia / mechanistic inhibition / OXIDOREDUCTASE
Function / homology
Function and homology information


ribonucleoside diphosphate metabolic process / 2'-deoxyribonucleotide biosynthetic process / nucleobase-containing small molecule interconversion / ribonucleoside-diphosphate reductase complex / ribonucleoside-diphosphate reductase / ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor / deoxyribonucleotide biosynthetic process / protein folding chaperone / iron ion binding / ATP binding ...ribonucleoside diphosphate metabolic process / 2'-deoxyribonucleotide biosynthetic process / nucleobase-containing small molecule interconversion / ribonucleoside-diphosphate reductase complex / ribonucleoside-diphosphate reductase / ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor / deoxyribonucleotide biosynthetic process / protein folding chaperone / iron ion binding / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
ATP-cone domain / ATP cone domain / ATP-cone domain profile. / Ribonucleotide reductase, class I , alpha subunit / Ribonucleotide reductase large subunit signature. / Ribonucleoside-diphosphate reductase large subunit / Ribonucleotide reductase R1 subunit, N-terminal / Ribonucleotide reductase large subunit, N-terminal / Ribonucleotide reductase, all-alpha domain / Ribonucleotide reductase large subunit, C-terminal ...ATP-cone domain / ATP cone domain / ATP-cone domain profile. / Ribonucleotide reductase, class I , alpha subunit / Ribonucleotide reductase large subunit signature. / Ribonucleoside-diphosphate reductase large subunit / Ribonucleotide reductase R1 subunit, N-terminal / Ribonucleotide reductase large subunit, N-terminal / Ribonucleotide reductase, all-alpha domain / Ribonucleotide reductase large subunit, C-terminal / Ribonucleotide reductase, barrel domain / Ribonucleotide reductase small subunit, acitve site / Ribonucleotide reductase small subunit signature. / Ribonucleotide reductase small subunit / Ribonucleotide reductase small subunit family / Ribonucleotide reductase, small chain / Ribonucleotide reductase-like / Ferritin-like superfamily
Similarity search - Domain/homology
Ribonucleoside-diphosphate reductase 1 subunit alpha / Ribonucleoside-diphosphate reductase 1 subunit beta
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsWestmoreland DE / Drennan CL
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R35GM126982-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM047274 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM29595 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1F32GM145072-01 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: 2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor NCDP.
Authors: Dana E Westmoreland / Patricia R Feliciano / Gyunghoon Kang / Chang Cui / Albert Kim / JoAnne Stubbe / Daniel G Nocera / Catherine L Drennan /
Abstract: Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the ...Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2β2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species. Here, we use cryogenic electron microscopy and a mechanism-based inhibitor 2'-azido-2'-deoxycytidine-5'-diphosphate (NCDP) to trap a wild-type α2β2 complex of class Ia RNR. We find that one α subunit has turned over and that the other is trapped, bound to β in a midturnover state. Instead of NCDP in the active site, forward RT has resulted in N loss, migration of the third nitrogen from the ribose C2' to C3' positions, and attachment of this nitrogen to the sulfur of cysteine-225. In this study, an inhibitor has been visualized as an adduct to an RNR. Additionally, this structure reveals the positions of PCET residues following forward RT, complementing the previous structure that depicted a preturnover PCET pathway and suggesting how PCET is gated at the α-β interface. This NCDP-trapped structure is also of sufficient resolution (2.6 Å) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process.
History
DepositionAug 23, 2024-
Header (metadata) releaseNov 6, 2024-
Map releaseNov 6, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46711.png

Downloads & links

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Map

FileDownload / File: emd_46711.map.gz / Format: CCP4 / Size: 729 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal map following density modification
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.42 Å/pix.
x 576 pix.
= 239.04 Å		0.42 Å/pix.
x 576 pix.
= 239.04 Å		0.42 Å/pix.
x 576 pix.
= 239.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.415 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.235
Minimum - Maximum-2.5624342 - 5.2813964
Average (Standard dev.)0.0000418912 (±0.1406615)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions576576576
Spacing576576576
CellA=B=C: 239.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_46711_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_46711_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_46711_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Active state of class Ia ribonucleotide reductase trapped with me...

EntireName: Active state of class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP
Components
  • Complex: Active state of class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP
    • Protein or peptide: Ribonucleoside-diphosphate reductase 1 subunit alpha
    • Protein or peptide: Ribonucleoside-diphosphate reductase 1 subunit beta
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: UNKNOWN LIGAND
  • Ligand: 4-amino-1-{(3xi)-3-C-amino-2-deoxy-5-O-[(S)-hydroxy(phosphonooxy)phosphoryl]-beta-D-threo-pentofuranosyl}pyrimidin-2(1H)-one
  • Ligand: MU-OXO-DIIRON
  • Ligand: water

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Supramolecule #1: Active state of class Ia ribonucleotide reductase trapped with me...

SupramoleculeName: Active state of class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 260.69 KDa

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Macromolecule #1: Ribonucleoside-diphosphate reductase 1 subunit alpha

MacromoleculeName: Ribonucleoside-diphosphate reductase 1 subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: ribonucleoside-diphosphate reductase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 85.877086 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNQNLLVTKR DGSTERINLD KIHRVLDWAA EGLHNVSISQ VELRSHIQFY DGIKTSDIHE TIIKAAADLI SRDAPDYQYL AARLAIFHL RKKAYGQFEP PALYDHVVKM VEMGKYDNHL LEDYTEEEFK QMDTFIDHDR DMTFSYAAVK QLEGKYLVQN R VTGEIYES ...String:
MNQNLLVTKR DGSTERINLD KIHRVLDWAA EGLHNVSISQ VELRSHIQFY DGIKTSDIHE TIIKAAADLI SRDAPDYQYL AARLAIFHL RKKAYGQFEP PALYDHVVKM VEMGKYDNHL LEDYTEEEFK QMDTFIDHDR DMTFSYAAVK QLEGKYLVQN R VTGEIYES AQFLYILVAA CLFSNYPRET RLQYVKRFYD AVSTFKISLP TPIMSGVRTP TRQFSSCVLI ECGDSLDSIN AT SSAIVKY VSQRAGIGIN AGRIRALGSP IRGGEAFHTG CIPFYKHFQT AVKSCSQGGV RGGAATLFYP MWHLEVESLL VLK NNRGVE GNRVRHMDYG VQINKLMYTR LLKGEDITLF SPSDVPGLYD AFFADQEEFE RLYTKYEKDD SIRKQRVKAV ELFS LMMQE RASTGRIYIQ NVDHCNTHSP FDPAIAPVRQ SNLCLEIALP TKPLNDVNDE NGEIALCTLS AFNLGAINNL DELEE LAIL AVRALDALLD YQDYPIPAAK RGAMGRRTLG IGVINFAYYL AKHGKRYSDG SANNLTHKTF EAIQYYLLKA SNELAK EQG ACPWFNETTY AKGILPIDTY KKDLDTIANE PLHYDWEALR ESIKTHGLRN STLSALMPSE TSSQISNATN GIEPPRG YV SIKASKDGIL RQVVPDYEHL HDAYELLWEM PGNDGYLQLV GIMQKFIDQS ISANTNYDPS RFPSGKVPMQ QLLKDLLT A YKFGVKTLYY QNTRDGAEDA QDDLVPSIQD DGCESGACKI

UniProtKB: Ribonucleoside-diphosphate reductase 1 subunit alpha

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Macromolecule #2: Ribonucleoside-diphosphate reductase 1 subunit beta

MacromoleculeName: Ribonucleoside-diphosphate reductase 1 subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: ribonucleoside-diphosphate reductase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 43.558055 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAYTTFSQTK NDQLKEPMFF GQPVNVARYD QQKYDIFEKL IEKQLSFFWR PEEVDVSRDR IDYQALPEHE KHIFISNLKY QTLLDSIQG RSPNVALLPL ISIPELETWV ETWAFSETIH SRSYTHIIRN IVNDPSVVFD DIVTNEQIQK RAEGISSYYD E LIEMTSYW ...String:
MAYTTFSQTK NDQLKEPMFF GQPVNVARYD QQKYDIFEKL IEKQLSFFWR PEEVDVSRDR IDYQALPEHE KHIFISNLKY QTLLDSIQG RSPNVALLPL ISIPELETWV ETWAFSETIH SRSYTHIIRN IVNDPSVVFD DIVTNEQIQK RAEGISSYYD E LIEMTSYW HLLGEGTHTV NGKTVTVSLR ELKKKLYLCL MSVNALEAIR FYVSFACSFA FAERELMEGN AKIIRLIARD EA LHLTGTQ HMLNLLRSGA DDPEMAEIAE ECKQECYDLF VQAAQQEKDW ADYLFRDGSM IGLNKDILCQ YVEYITNIRM QAV GLDLPF QTRSNPIPWI NTWLVSDNVQ VAPQEVEVSS YLVGQIDSEV DTDDLSNFQL

UniProtKB: Ribonucleoside-diphosphate reductase 1 subunit beta

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Macromolecule #3: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

ChemComp-DTP:
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 4 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #6: UNKNOWN LIGAND

MacromoleculeName: UNKNOWN LIGAND / type: ligand / ID: 6 / Number of copies: 1 / Formula: UNL
Molecular weightTheoretical: 214.11 Da
Chemical component information


ChemComp, No image

ChemComp-UNL:
Unknown ligand

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Macromolecule #7: 4-amino-1-{(3xi)-3-C-amino-2-deoxy-5-O-[(S)-hydroxy(phosphonooxy)...

MacromoleculeName: 4-amino-1-{(3xi)-3-C-amino-2-deoxy-5-O-[(S)-hydroxy(phosphonooxy)phosphoryl]-beta-D-threo-pentofuranosyl}pyrimidin-2(1H)-one
type: ligand / ID: 7 / Number of copies: 1 / Formula: A1A3L
Molecular weightTheoretical: 402.192 Da

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Macromolecule #8: MU-OXO-DIIRON

MacromoleculeName: MU-OXO-DIIRON / type: ligand / ID: 8 / Number of copies: 2 / Formula: FEO
Molecular weightTheoretical: 127.689 Da
Chemical component information

ChemComp-FEO:
MU-OXO-DIIRON

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 624 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.946 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 440549
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Details: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Details: RELION
FSC plot (resolution estimation)

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