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- EMDB-32782: Cryo-EM structure of human pyruvate carboxylase with acetyl-CoA i... -

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Basic information

Entry
Database: EMDB / ID: EMD-32782
TitleCryo-EM structure of human pyruvate carboxylase with acetyl-CoA in the intermediate state 4
Map data
Sample
  • Complex: PC
    • Protein or peptide: Pyruvate carboxylase
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsChai P / Lan P / Wu J / Lei M
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Mol Cell / Year: 2022
Title: Mechanistic insight into allosteric activation of human pyruvate carboxylase by acetyl-CoA.
Authors: Peiwei Chai / Pengfei Lan / Shaobai Li / Deqiang Yao / Chenchen Chang / Mi Cao / Yafeng Shen / Shengfang Ge / Jian Wu / Ming Lei / Xianqun Fan /
Abstract: Pyruvate carboxylase (PC) catalyzes the two-step carboxylation of pyruvate to produce oxaloacetate, playing a key role in the maintenance of metabolic homeostasis in cells. Given its involvement in ...Pyruvate carboxylase (PC) catalyzes the two-step carboxylation of pyruvate to produce oxaloacetate, playing a key role in the maintenance of metabolic homeostasis in cells. Given its involvement in multiple diseases, PC has been regarded as a potential therapeutic target for obesity, diabetes, and cancer. Albeit acetyl-CoA has been recognized as the allosteric regulator of PC for over 60 years, the underlying mechanism of how acetyl-CoA induces PC activation remains enigmatic. Herein, by using time-resolved cryo-electron microscopy, we have captured the snapshots of PC transitional states during its catalytic cycle. These structures and the biochemical studies reveal that acetyl-CoA stabilizes PC in a catalytically competent conformation, which triggers a cascade of events, including ATP hydrolysis and the long-distance communication between the two reactive centers. These findings provide an integrated picture for PC catalysis and unveil the unique allosteric mechanism of acetyl-CoA in an essential biochemical reaction in all kingdoms of life.
History
DepositionFeb 4, 2022-
Header (metadata) releaseNov 9, 2022-
Map releaseNov 9, 2022-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32782.png

Downloads & links

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Map

FileDownload / File: emd_32782.map.gz / Format: CCP4 / Size: 115.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 312 pix.
= 343.2 Å		1.1 Å/pix.
x 312 pix.
= 343.2 Å		1.1 Å/pix.
x 312 pix.
= 343.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.015063928 - 0.034503236
Average (Standard dev.)0.000102614285 (±0.0014561766)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions312312312
Spacing312312312
CellA=B=C: 343.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : PC

EntireName: PC
Components
  • Complex: PC
    • Protein or peptide: Pyruvate carboxylase

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Supramolecule #1: PC

SupramoleculeName: PC / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Pyruvate carboxylase

MacromoleculeName: Pyruvate carboxylase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MLKFRTVHGG LRLLGIRRTS TAPAASPNVR RLEYKPIKKV MVANRGEIAI RVFRACTELG IRTVAIYSEQ DTGQMHRQK ADEAYLIGRG LAPVQAYLHI PDIIKVAKEN NVDAVHPGYG FLSERADFAQ ACQDAGVRFI G PSPEVVRK MGDKVEARAI AIAAGVPVVP ...String:
MLKFRTVHGG LRLLGIRRTS TAPAASPNVR RLEYKPIKKV MVANRGEIAI RVFRACTELG IRTVAIYSEQ DTGQMHRQK ADEAYLIGRG LAPVQAYLHI PDIIKVAKEN NVDAVHPGYG FLSERADFAQ ACQDAGVRFI G PSPEVVRK MGDKVEARAI AIAAGVPVVP GTDAPITSLH EAHEFSNTYG FPIIFKAAYG GGGRGMRVVH SY EELEENY TRAYSEALAA FGNGALFVEK FIEKPRHIEV QILGDQYGNI LHLYERDCSI QRRHQKVVEI APA AHLDPQ LRTRLTSDSV KLAKQVGYEN AGTVEFLVDR HGKHYFIEVN SRLQVEHTVT EEITDVDLVH AQIH VAEGR SLPDLGLRQE NIRINGCAIQ CRVTTEDPAR SFQPDTGRIE VFRSGEGMGI RLDNASAFQG AVISP HYDS LLVKVIAHGK DHPTAATKMS RALAEFRVRG VKTNIAFLQN VLNNQQFLAG TVDTQFIDEN PELFQL RPA QNRAQKLLHY LGHVMVNGPT TPIPVKASPS PTDPVVPAVP IGPPPAGFRD ILLREGPEGF ARAVRNH PG LLLMDTTFRD AHQSLLATRV RTHDLKKIAP YVAHNFSKLF SMENWGGATF DVAMRFLYEC PWRRLQEL R ELIPNIPFQM LLRGANAVGY TNYPDNVVFK FCEVAKENGM DVFRVFDSLN YLPNMLLGME AAGSAGGVV EAAISYTGDV ADPSRTKYSL QYYMGLAEEL VRAGTHILCI KDMAGLLKPT ACTMLVSSLR DRFPDLPLHI HTHDTSGAG VAAMLACAQA GADVVDVAAD SMSGMTSQPS MGALVACTRG TPLDTEVPME RVFDYSEYWE G ARGLYAAF DCTATMKSGN SDVYENEIPG GQYTNLHFQA HSMGLGSKFK EVKKAYVEAN QMLGDLIKVT PS SKIVGDL AQFMVQNGLS RAEAEAQAEE LSFPRSVVEF LQGYIGVPHG GFPEPFRSKV LKDLPRVEGR PGA SLPPLD LQALEKELVD RHGEEVTPED VLSAAMYPDV FAHFKDFTAT FGPLDSLNTR LFLQGPKIAE EFEV ELERG KTLHIKALAV SDLNRAGQRQ VFFELNGQLR SILVKDTQAM KEMHFHPKAL KDVKGQIGAP MPGKV IDIK VVAGAKVAKG QPLCVLSAMK METVVTSPME GTVRKVHVTK DMTLEGDDLI LEIE

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3bg5

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 74361
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: MAXIMUM LIKELIHOOD

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