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- EMDB-60263: Cryo-EM structure of R-eLACCO2 in the lactate-bound state -

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Basic information

Entry
Database: EMDB / ID: EMD-60263
TitleCryo-EM structure of R-eLACCO2 in the lactate-bound state
Map data
Sample
  • Complex: R-eLACCO2 in the lactate-bound state
    • Protein or peptide: Lactate-binding periplasmic protein TTHA0766,Red fluorescent protein drFP583
  • Ligand: CALCIUM ION
  • Ligand: (2S)-2-HYDROXYPROPANOIC ACID
KeywordsFLUORESCENT PROTEIN
Function / homology
Function and homology information


lactate transport / tripartite ATP-independent periplasmic transporter complex / bioluminescence / generation of precursor metabolites and energy / transmembrane transport / periplasmic space / calcium ion binding / protein homodimerization activity / zinc ion binding
Similarity search - Function
Lactate-binding periplasmic protein TTHA0766 / Solute binding protein, TakP-like / TRAP transporter solute receptor DctP / TRAP transporter solute receptor DctP superfamily / Bacterial extracellular solute-binding protein, family 7 / Green fluorescent protein, GFP / Twin arginine translocation (Tat) signal profile. / Twin-arginine translocation pathway, signal sequence / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
Lactate-binding periplasmic protein TTHA0766 / Red fluorescent protein drFP583
Similarity search - Component
Biological speciesThermus thermophilus H8 (bacteria) / Thermus thermophilus HB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsKamijo Y / Kusakizako T / Nureki O / Campbell RE / Nasu Y
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
Japan Science and Technology Japan
CitationJournal: Nat Commun / Year: 2025
Title: A red fluorescent genetically encoded biosensor for in vivo imaging of extracellular L-lactate dynamics.
Authors: Yuki Kamijo / Philipp Mächler / Natalie Ness / Cong Quang Vu / Tsukasa Kusakizako / Jamsad Mannuthodikayil / Zaneta Ku / Marc Boisvert / Ekaterina Grebenik / Ikumi Miyazaki / Rina Hashizume ...Authors: Yuki Kamijo / Philipp Mächler / Natalie Ness / Cong Quang Vu / Tsukasa Kusakizako / Jamsad Mannuthodikayil / Zaneta Ku / Marc Boisvert / Ekaterina Grebenik / Ikumi Miyazaki / Rina Hashizume / Haruaki Sato / Rui Liu / Yukiko Hori / Taisuke Tomita / Tetsuro Katayama / Akihiro Furube / Gabriela Caraveo / Marie-Eve Paquet / Mikhail Drobizhev / Osamu Nureki / Satoshi Arai / Marco Brancaccio / Robert E Campbell / David Kleinfeld / Yusuke Nasu /
Abstract: L-Lactate is increasingly recognized as an intercellular energy currency in mammals, but mysteries remain regarding the spatial and temporal dynamics of its release and uptake between cells via the ...L-Lactate is increasingly recognized as an intercellular energy currency in mammals, but mysteries remain regarding the spatial and temporal dynamics of its release and uptake between cells via the extracellular environment. Here we introduce R-eLACCO2.1, a red fluorescent extracellular L-lactate biosensor that is superior to previously reported green fluorescent biosensors in in vivo sensitivity to increases in extracellular L-lactate and spectral orthogonality. R-eLACCO2.1 exhibits excellent fluorescence response in cultured cells, mouse brain slices, and live mice. R-eLACCO2.1 also serves as an effective fluorescence lifetime-based biosensor. Using R-eLACCO2.1, we monitor whisker stimulation and locomotion-induced changes in endogenous extracellular L-lactate in the somatosensory cortex of awake mice. To highlight the potential insights gained from in vivo measurements with R-eLACCO2.1, we perform dual-color imaging from the somatosensory cortex of actively locomoting mice. This enables us to simultaneously observe the neural activity, reported by a green fluorescent GCaMP calcium ion biosensor, and extracellular L-lactate. As the high-performance tool in the suite of extracellular L-lactate biosensors, R-eLACCO2.1 is ideally suited to delimit the emerging roles of L-lactate in mammalian metabolism.
History
DepositionMay 24, 2024-
Header (metadata) releaseOct 29, 2025-
Map releaseOct 29, 2025-
UpdateNov 12, 2025-
Current statusNov 12, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60263.png

Downloads & links

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Map

FileDownload / File: emd_60263.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.13539685 - 0.2619354
Average (Standard dev.)-0.00020225433 (±0.0042164717)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_60263_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_60263_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : R-eLACCO2 in the lactate-bound state

EntireName: R-eLACCO2 in the lactate-bound state
Components
  • Complex: R-eLACCO2 in the lactate-bound state
    • Protein or peptide: Lactate-binding periplasmic protein TTHA0766,Red fluorescent protein drFP583
  • Ligand: CALCIUM ION
  • Ligand: (2S)-2-HYDROXYPROPANOIC ACID

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Supramolecule #1: R-eLACCO2 in the lactate-bound state

SupramoleculeName: R-eLACCO2 in the lactate-bound state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermus thermophilus H8 (bacteria)

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Macromolecule #1: Lactate-binding periplasmic protein TTHA0766,Red fluorescent prot...

MacromoleculeName: Lactate-binding periplasmic protein TTHA0766,Red fluorescent protein drFP583
type: protein_or_peptide / ID: 1 / Details: Chimeric protein of THA0766 and cpmApple / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 70.0445 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDPSSRMFSP MAVAQARRYK WRIQTAWDAG TVGYSLFQKF TERVKELTDG QLEVQPLPA GAVVGTFDMF DAVKTGVLDG MNPFTLYWAE RMPVTAFLSS YALGLDRPDQ WETWFYSLGG LDNARRAFAE Q GLFYVGPV ...String:
MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDPSSRMFSP MAVAQARRYK WRIQTAWDAG TVGYSLFQKF TERVKELTDG QLEVQPLPA GAVVGTFDMF DAVKTGVLDG MNPFTLYWAE RMPVTAFLSS YALGLDRPDQ WETWFYSLGG LDNARRAFAE Q GLFYVGPV QHDLNTIHSK KPIRRFEDFK GVKLRVPGGM IAEVFAAAGA STVLLPGGEV YPALERGAVT AVSERMYPED GA LKSEIKK GLRLKDGGHY AAVVKTTYKA KKPVQLPGAY VVDIQLDIVS HNEDYTIVEQ CERAEGRHST GGVVELDKGG TGG SLVSKG EEDDMAIVKE FMRFKVHMEG SVNGHEFEIE GEGEGRPYEA FQTAKLKVTK GGPLPFAWDI LSPQF(NRQ)SKAY IKHPADIPD YFKLSFPEGF RWERVMYFED GGIIHVDQDS SLQDGVFIYK VKLRGTNFPP DGPVMQKKTM GWERGRSAAD F VGPAVNYN LGFHQEAKYI IMGPPETPAI HQPVDLMDFT INLNRWRSLP KPLQERFIAA VHEYSWIHYA GIQKANLEAW PK YRQAGVE VIRLGNEDVR KFRRLAIPIW FKWAKMDKYS REAFASQLEY MRGIGHVTDE ELKGLSL

UniProtKB: Lactate-binding periplasmic protein TTHA0766, Red fluorescent protein drFP583, Red fluorescent protein drFP583, Lactate-binding periplasmic protein TTHA0766

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: (2S)-2-HYDROXYPROPANOIC ACID

MacromoleculeName: (2S)-2-HYDROXYPROPANOIC ACID / type: ligand / ID: 3 / Number of copies: 1 / Formula: 2OP
Molecular weightTheoretical: 90.078 Da
Chemical component information

ChemComp-2OP:
(2S)-2-HYDROXYPROPANOIC ACID

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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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup model#0 - Type of model: OTHER / #1 - Type of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 146454
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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