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- EMDB-19601: GRP94-BiP complex, loading conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-19601
TitleGRP94-BiP complex, loading conformation
Map datarefined, sharpened map of GRP94-BiP-HT2 complex in the loading conformation, negative-stain EM data
Sample
  • Complex: Recombinant complex of human GRP94, BiP, and a Halo-Tag 2 (HT2) substrate
    • Complex: GRP94
      • Protein or peptide: GRP94 delta1-72, N-term Strep-tag
    • Complex: BiP
      • Protein or peptide: BiP, His-tagged
    • Complex: Halo-Tag 2
      • Protein or peptide: HaloTag2, N-term Spot-Tag
Keywordschaperone / HSP90 / co-chaperone / HSP70
Function / homology
Function and homology information


positive regulation of toll-like receptor signaling pathway / actin rod assembly / : / regulation of ATF6-mediated unfolded protein response / regulation of PERK-mediated unfolded protein response / regulation of protein folding in endoplasmic reticulum / cerebellum structural organization / ATF6 (ATF6-alpha) activates chaperones / ATF6B (ATF6-beta) activates chaperones / maintenance of protein localization in endoplasmic reticulum ...positive regulation of toll-like receptor signaling pathway / actin rod assembly / : / regulation of ATF6-mediated unfolded protein response / regulation of PERK-mediated unfolded protein response / regulation of protein folding in endoplasmic reticulum / cerebellum structural organization / ATF6 (ATF6-alpha) activates chaperones / ATF6B (ATF6-beta) activates chaperones / maintenance of protein localization in endoplasmic reticulum / IRE1alpha activates chaperones / ATF6 (ATF6-alpha) activates chaperone genes / endoplasmic reticulum chaperone complex / negative regulation of IRE1-mediated unfolded protein response / regulation of IRE1-mediated unfolded protein response / PERK regulates gene expression / sarcoplasmic reticulum lumen / protein folding in endoplasmic reticulum / post-translational protein targeting to membrane, translocation / cerebellar Purkinje cell layer development / misfolded protein binding / Trafficking and processing of endosomal TLR / Modulation of host responses by IFN-stimulated genes / Scavenging by Class A Receptors / low-density lipoprotein particle receptor binding / retrograde protein transport, ER to cytosol / protein phosphatase inhibitor activity / IRE1-mediated unfolded protein response / negative regulation of PERK-mediated unfolded protein response / endoplasmic reticulum-Golgi intermediate compartment / smooth endoplasmic reticulum / cellular response to ATP / non-chaperonin molecular chaperone ATPase / intracellular membrane-bounded organelle / Regulation of HSF1-mediated heat shock response / protein serine/threonine kinase inhibitor activity / Dengue Virus Attachment and Entry / positive regulation of Wnt signaling pathway / negative regulation of protein-containing complex assembly / cellular response to glucose starvation / cellular response to manganese ion / endoplasmic reticulum unfolded protein response / heat shock protein binding / endocytic vesicle lumen / ERAD pathway / protein folding chaperone / substantia nigra development / response to endoplasmic reticulum stress / protein localization to plasma membrane / positive regulation of protein ubiquitination / ATP-dependent protein folding chaperone / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / Post-translational protein phosphorylation / Maturation of DENV proteins / protein sequestering activity / protein refolding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / : / melanosome / Platelet degranulation / protein transport / protein-folding chaperone binding / ribosome binding / Interleukin-4 and Interleukin-13 signaling / protein folding / midbody / protein phosphatase binding / response to hypoxia / positive regulation of cell migration / cadherin binding / endoplasmic reticulum lumen / protein domain specific binding / focal adhesion / ubiquitin protein ligase binding / calcium ion binding / negative regulation of apoptotic process / endoplasmic reticulum membrane / perinuclear region of cytoplasm / enzyme binding / cell surface / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / protein-containing complex / mitochondrion / RNA binding / extracellular exosome / extracellular region / ATP binding / membrane / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Endoplasmic reticulum chaperone BIP, nucleotide-binding domain / Endoplasmic reticulum targeting sequence. / Heat shock hsp70 proteins family signature 2. / Heat shock hsp70 proteins family signature 1. / Heat shock hsp70 proteins family signature 3. / Heat shock protein 70, conserved site / Heat shock protein 70kD, peptide-binding domain superfamily / Heat shock protein 70kD, C-terminal domain superfamily / Heat shock protein 70 family / Hsp70 protein ...Endoplasmic reticulum chaperone BIP, nucleotide-binding domain / Endoplasmic reticulum targeting sequence. / Heat shock hsp70 proteins family signature 2. / Heat shock hsp70 proteins family signature 1. / Heat shock hsp70 proteins family signature 3. / Heat shock protein 70, conserved site / Heat shock protein 70kD, peptide-binding domain superfamily / Heat shock protein 70kD, C-terminal domain superfamily / Heat shock protein 70 family / Hsp70 protein / Heat shock protein Hsp90, conserved site / Heat shock hsp90 proteins family signature. / HSP90, C-terminal domain / Heat shock protein Hsp90, N-terminal / Heat shock protein Hsp90 family / Hsp90 protein / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / ATPase, nucleotide binding domain / Ribosomal protein S5 domain 2-type fold
Similarity search - Domain/homology
Endoplasmic reticulum chaperone BiP / Endoplasmin
Similarity search - Component
Biological speciesHomo sapiens (human) / Rhodococcus (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 14.0 Å
AuthorsBrenner JC / Zirden LC / Almeida-Hernandez Y / Kaschani F / Kaiser M / Sanchez-Garcia E / Poepsel S / Hellerschmied D
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)424228829 Germany
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Conformational plasticity of a BiP-GRP94 chaperone complex.
Authors: Joel Cyrille Brenner / Linda Charlotte Zirden / Lana Buzuk / Yasser Almeida-Hernandez / Lea Radzuweit / Joao Diamantino / Farnusch Kaschani / Markus Kaiser / Elsa Sanchez-Garcia / Simon ...Authors: Joel Cyrille Brenner / Linda Charlotte Zirden / Lana Buzuk / Yasser Almeida-Hernandez / Lea Radzuweit / Joao Diamantino / Farnusch Kaschani / Markus Kaiser / Elsa Sanchez-Garcia / Simon Poepsel / Doris Hellerschmied /
Abstract: Hsp70 and Hsp90 chaperones and their regulatory cochaperones are critical for maintaining protein homeostasis. Glucose-regulated protein 94 (GRP94), the sole Hsp90 chaperone in the secretory pathway ...Hsp70 and Hsp90 chaperones and their regulatory cochaperones are critical for maintaining protein homeostasis. Glucose-regulated protein 94 (GRP94), the sole Hsp90 chaperone in the secretory pathway of mammalian cells, is essential for the maturation of important secretory and transmembrane proteins. Without the requirement of cochaperones, the Hsp70 protein BiP controls regulatory conformational changes of GRP94, the structural basis of which has remained elusive. Here we biochemically and structurally characterize the formation of a BiP-GRP94 chaperone complex and its transition to a conformation expected to support the loading of substrate proteins from BiP onto GRP94. BiP initially binds to the open GRP94 dimer through an interaction interface that is conserved among Hsp70 and Hsp90 paralogs. Subsequently, binding of a second BiP protein stabilizes a semiclosed GRP94 dimer, thereby advancing the chaperone cycle. Our findings highlight a fundamental mechanism of direct Hsp70-Hsp90 cooperation, independent of cochaperones.
History
DepositionFeb 11, 2024-
Header (metadata) releaseJul 30, 2025-
Map releaseJul 30, 2025-
UpdateOct 22, 2025-
Current statusOct 22, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19601.png

Downloads & links

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Map

FileDownload / File: emd_19601.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationrefined, sharpened map of GRP94-BiP-HT2 complex in the loading conformation, negative-stain EM data
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.89 Å/pix.
x 200 pix.
= 378. Å		1.89 Å/pix.
x 200 pix.
= 378. Å		1.89 Å/pix.
x 200 pix.
= 378. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.89 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.38
Minimum - Maximum-0.32166648 - 3.1107554
Average (Standard dev.)0.0032266465 (±0.18321009)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 378.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened map of GRP94-BiP-HT2 complex in the loading...

Fileemd_19601_additional_1.map
Annotationunsharpened map of GRP94-BiP-HT2 complex in the loading conformation, negative-stain EM data
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: refined, sharpened map of GRP94-BiP-HT2 complex in the...

Fileemd_19601_additional_2.map
Annotationrefined, sharpened map of GRP94-BiP-HT2 complex in the loading conformation, negative-stain EM data, as obtained from heterogeneous refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_19601_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_19601_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Recombinant complex of human GRP94, BiP, and a Halo-Tag 2 (HT2) s...

EntireName: Recombinant complex of human GRP94, BiP, and a Halo-Tag 2 (HT2) substrate
Components
  • Complex: Recombinant complex of human GRP94, BiP, and a Halo-Tag 2 (HT2) substrate
    • Complex: GRP94
      • Protein or peptide: GRP94 delta1-72, N-term Strep-tag
    • Complex: BiP
      • Protein or peptide: BiP, His-tagged
    • Complex: Halo-Tag 2
      • Protein or peptide: HaloTag2, N-term Spot-Tag

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Supramolecule #1: Recombinant complex of human GRP94, BiP, and a Halo-Tag 2 (HT2) s...

SupramoleculeName: Recombinant complex of human GRP94, BiP, and a Halo-Tag 2 (HT2) substrate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Complex was reconstituted from individual purified proteins and then purified and fixated by glycerol gradient ultracentrifugation with glutaraldehyde cross-linking (GraFix)
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35 KDa

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Supramolecule #2: GRP94

SupramoleculeName: GRP94 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: BiP

SupramoleculeName: BiP / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Halo-Tag 2

SupramoleculeName: Halo-Tag 2 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Rhodococcus (bacteria)

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Macromolecule #1: GRP94 delta1-72, N-term Strep-tag

MacromoleculeName: GRP94 delta1-72, N-term Strep-tag / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASWSHPQFE KGSMGKPIPN PLLGLDSTEN LYFQGKSEKF AFQAEVNRMM KLIINSLYKN KEIFLRELIS NASDALDKIR LISLTDENAL AGNEELTVKI KCDKEKNLLH VTDTGVGMTR EELVKNLGTI AKSGTSEFLN KMTEAQEDGQ STSELIGQFG VGFYSAFLVA ...String:
MASWSHPQFE KGSMGKPIPN PLLGLDSTEN LYFQGKSEKF AFQAEVNRMM KLIINSLYKN KEIFLRELIS NASDALDKIR LISLTDENAL AGNEELTVKI KCDKEKNLLH VTDTGVGMTR EELVKNLGTI AKSGTSEFLN KMTEAQEDGQ STSELIGQFG VGFYSAFLVA DKVIVTSKHN NDTQHIWESD SNEFSVIADP RGNTLGRGTT ITLVLKEEAS DYLELDTIKN LVRKYSQFIN FPIYVWSSKT ETVEEPLEED EAAKEEKEES DDEAAVEEEE EEKKPKTKKV EKTVWDWELM NDIKPIWQRP SKEVEEDEYK AFYKSFSKES DDPMAYIHFT AEGEVTFKSI LFVPTSAPRG LFDEYGSKKS DYIKLYVRRV FITDDFHDMM PKYLNFVKGV VDSDDLPLNV SRETLQQHKL LKVIRKKLVR KTLDMIKKIA DEKYNDTFWK EFGTNIKLGV IEDHSNRTRL AKLLRFQSSH HSTDITSLDQ YVERMKEKQD KIYFMAGSSR KEAESSPFVE RLLKKGYEVI YLTEPVDEYC IQALPEFDGK RFQNVAKEGV KFDESEKTKE SREATEKEFE PLLNWMKDKA LKDKIEKAVV SQRLTESPCA LVASQYGWSG NMERIMKAQA YQTGKDISTN YYASQKKTFE INPRHPLIRD MLRRIKEDED DKTVMDLAVV LFETATLRSG YLLPDTKAYG DRIERMLRLS LNIDPEAQVE EEPEEEPEDT SEEAEDSEQD EGEEMDAGTE EEEEETEKES TEKDEL

UniProtKB: Endoplasmin

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Macromolecule #2: BiP, His-tagged

MacromoleculeName: BiP, His-tagged / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHSSG VDLGTENLYF QSMDVGTVVG IDLGTTYSCV GVFKNGRVEI IANDQGNRIT PSYVAFTPEG ERLIGDAAKN QLTSNPENTV FDAKRLIGRT WNDPSVQQDI KFLPFKVVEK KTKPYIQVDI GGGQTKTFAP EEISAMVLTK MKETAEAYLG KKVTHAVVTV ...String:
MHHHHHHSSG VDLGTENLYF QSMDVGTVVG IDLGTTYSCV GVFKNGRVEI IANDQGNRIT PSYVAFTPEG ERLIGDAAKN QLTSNPENTV FDAKRLIGRT WNDPSVQQDI KFLPFKVVEK KTKPYIQVDI GGGQTKTFAP EEISAMVLTK MKETAEAYLG KKVTHAVVTV PAYFNDAQRQ ATKDAGTIAG LNVMRIINEP TAAAIAYGLD KREGEKNILV FDLGGGTFDV SLLTIDNGVF EVVATNGDTH LGGEDFDQRV MEHFIKLYKK KTGKDVRKDN RAVQKLRREV EKAKRALSSQ HQARIEIESF FEGEDFSETL TRAKFEELNM DLFRSTMKPV QKVLEDSDLK KSDIDEIVLV GGSTRIPKIQ QLVKEFFNGK EPSRGINPDE AVAYGAAVQA GVLSGDQDTG DLVLLDVCPL TLGIETVGGV MTKLIPRNTV VPTKKSQIFS TASDNQPTVT IKVYEGERPL TKDNHLLGTF DLTGIPPAPR GVPQIEVTFE IDVNGILRVT AEDKGTGNKN KITITNDQNR LTPEEIERMV NDAEKFAEED KKLKERIDTR NELESYAYSL KNQIGDKEKL GGKLSSEDKE TMEKAVEEKI EWLESHQDAD IEDFKAKKKE LEEIVQPIIS KLYGSGGPPP TGEEDTSEKD EL

UniProtKB: Endoplasmic reticulum chaperone BiP

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Macromolecule #3: HaloTag2, N-term Spot-Tag

MacromoleculeName: HaloTag2, N-term Spot-Tag / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MPDRVRAVSH WSSASSEIGT GFPFDPHYVE VLGERMHYVD VGPRDGTPVL FLHGNPTSSY LWRNIIPHVA PSHRCIAPDL IGMGKSDKPD LDYFFDDHVR YLDAFIEALG LEEVVLVIHD WGSALGFHWA KRNPERVKGI ACMEFIRPIP TWDEWPEFAR ETFQAFRTAD ...String:
MPDRVRAVSH WSSASSEIGT GFPFDPHYVE VLGERMHYVD VGPRDGTPVL FLHGNPTSSY LWRNIIPHVA PSHRCIAPDL IGMGKSDKPD LDYFFDDHVR YLDAFIEALG LEEVVLVIHD WGSALGFHWA KRNPERVKGI ACMEFIRPIP TWDEWPEFAR ETFQAFRTAD VGRELIIDQN AFIEGALPMG VVRPLTEVEM DHYREPFLKP VDREPLWRFP NELPIAGEPA NIVALVEAYM NWLHQSPVPK LLFWGTPGVL IPPAEAARLA ESLPNCKTVD IGPGLFLLQE DNPDLIGSEI ARWLPGLAG

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
StainingType: NEGATIVE / Material: Uranyl formate
Details: 4 ul of the GraFix fraction was applied to a Copper 400 Mesh grids with continuous carbon (Electron Microscopy Sciences) after glow discharge, incubated for 30 s, immediately blotted with ...Details: 4 ul of the GraFix fraction was applied to a Copper 400 Mesh grids with continuous carbon (Electron Microscopy Sciences) after glow discharge, incubated for 30 s, immediately blotted with filter paper and stained via five successive short incubations of 2% (w/v) uranyl formate (Science Services). The excess stain was removed with filter paper and the grids dried before imaging
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.

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Electron microscopy

MicroscopeTFS TALOS L120C
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 22.0 µm / Nominal defocus min: 7.0 µm
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos L120C / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 31838
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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