Structure of a GRP94 folding intermediate engaged with a CCDC134- and FKBP11-bound secretory translocon

2026-01-28 REL RCSB INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY INITIAL_RELEASE REPOSITORY EXPERIMENTAL_SUMMARY em_admin pdbx_database_related _em_admin.last_update EXPERIMENTAL_SUMMARY citation em_admin _citation.journal_volume _citation.page_first _citation.page_last _citation.year _em_admin.last_update DATABASE_REFERENCES citation em_admin _citation.journal_volume _citation.page_first _citation.page_last _citation.year _em_admin.last_update RCSB RCSB 2025-09-29 2025-11-19 2025-11-19 2026-01-28 National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) R35GM145374 United States Structure of a GRP94 folding intermediate engaged with a CCDC134- and FKBP11-bound secretory translocon Yamsek M Jha R Keenan RJ Ribosome, Membrane protein, Translocon, Transport, N-Glycosylation Yamsek M Ma M Jha R Wan Y Li Q Zhong F DeLong K Ji Z Rohatgi R Keenan RJ Structural basis of regulated N-glycosylation at the secretory translocon. Nature UK 649 777 784 2026 41261126 doi:10.1038/s41586-025-09756-8 1476-4687 0006 NATUAS Ma M Dubey R Jen A Pusapati GV Singal B Shishkova E Overmyer KA Cormier-Daire V Fedry J Aravind L Coon JJ Rohatgi R Regulated N-glycosylation controls chaperone function and receptor trafficking Science US 2024 39509507 doi:10.1038/s41592-020-0967-9 1095-9203 0353 SCIEAS EMD-49171 other EM volume 9ygy FULLOVERLAP EMDB EMD-72945 associated EM volume

EMDB EMD-49171 other EM volume Nascent GRP94 folding intermediate bound to a ribosome-translocon complex Nascent GRP94 folding intermediate bound to a ribosome-translocon complex 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Isolated by affinity purification via a Flag-tag on FKBP11

Homo sapiens Endoplasmic reticulum Translocon-associated protein subunit alpha Homo sapiens 0.032263219 1 LEVO MRLLPRLLLLLLLVFPATVLFRGGPRGLLAVAQDLTEDEETVEDSIIEDEDDEAEVEEDEPTDLVEDKEEEDVSGEPEAS PSADTTILFVKGEDFPANNIVKFLVGFTNKGTEDFIVESLDASFRYPQDYQFYIQNFTALPLNTVVPPQRQATFEYSFIP AEPMGGRPFGLVINLNYKDLNGNVFQDAVFNQTVTVIEREDGLDGETIFMYMFLAGLGLLVIVGLHQLLESRKRKRPIQK VEMGTSSQNDVDMSWIPQETLNQINKASPRRLPRKRAQKRSVGSDE P43307 Translocon-associated protein subunit beta Homo sapiens 0.020154986 1 LEVO MRLLSFVVLALFAVTQAEEGARLLASKSLLNRYAVEGRDLTLQYNIYNVGSSAALDVELSDDSFPPEDFGIVSGMLNVKW DRIAPASNVSHTVVLRPLKAGYFNFTSATITYLAQEDGPVVIGSTSAPGQGGILAQREFDRRFSPHFLDWAAFGVMTLPS IGIPLLLWYSSKRKYDTPKTKKN P43308 Translocon-associated protein subunit gamma Homo sapiens 0.021106464999999998 1 LEVO MAPKGSSKQQSEEDLLLQDFSRNLSAKSSALFFGNAFIVSAIPIWLYWRIWHMDLIQSAVLYSVMTLVSTYLVAFAYKNV KFVLKHKVAQKREDAVSKEVTRKLSEADNRKMSRKEKDERILWKKNEVADYEATTFSIFYNNTLFLVVVIVASFFILKNF NPTVNYILSISASSGLIALLSTGSK Q9UNL2 Translocon-associated protein subunit delta Homo sapiens 0.019015546999999997 1 LEVO MAAMASLGALALLLLSSLSRCSAEACLEPQITPSYYTTSDAVISTETVFIVEISLTCKNRVQNMALYADVGGKQFPVTRG QDVGRYQVSWSLDHKSAHAGTYEVRFFDEESYSLLRKAQRNNEDISIIPPLFTVSVDHRGTWNGPWVSTEVLAAAIGLVI YYLAFSAKSHIQA P51571 Peptidyl-prolyl cis-trans isomerase FKBP11 Homo sapiens 0.022213392999999998 1 LEVO MTLRPSLLPLHLLLLLLLSAAVCRAEAGLETESPVRTLQVETLVEPPEPCAEPAAFGDTLHIHYTGSLVDGRIIDTSLTR DPLVIELGQKQVIPGLEQSLLDMCVGEKRRAIIPSHLAYGKRGFPPSVPADAVVQYDVELIALIRANYWLKLVKGILPLV GMAMVPALLGLIGYHLYRKANRPKVSKKKLKEEKRNKSKKK Q9NYL4 5.2.1.8 Coiled-coil domain-containing protein 134 Homo sapiens 0.026598648 1 LEVO MDLLQFLAFLFVLLLSGMGATGTLRTSLDPSLEIYKKMFEVKRREQLLALKNLAQLNDIHQQYKILDVMLKGLFKVLEDS RTVLTAADVLPDGPFPQDEKLKDAFSHVVENTAFFGDVVLRFPRIVHYYFDHNSNWNLLIRWGISFCNQTGVFNQGPHSP ILSLMAQELGISEKDSNFQNPFKIDRTEFIPSTDPFQKALREEEKRRKKEEKRKEIRKGPRISRSQSEL Q9H6E4 Protein transport protein Sec61 subunit alpha isoform 1 Homo sapiens 0.052304406 1 LEVO MAIKFLEVIKPFCVILPEIQKPERKIQFKEKVLWTAITLFIFLVCCQIPLFGIMSSDSADPFYWMRVILASNRGTLMELG ISPIVTSGLIMQLLAGAKIIEVGDTPKDRALFNGAQKLFGMIITIGQSIVYVMTGMYGDPSEMGAGICLLITIQLFVAGL IVLLLDELLQKGYGLGSGISLFIATNICETIVWKAFSPTTVNTGRGMEFEGAIIALFHLLATRTDKVRALREAFYRQNLP NLMNLIATIFVFAVVIYFQGFRVDLPIKSARYRGQYNTYPIKLFYTSNIPIILQSALVSNLYVISQMLSARFSGNLLVSL LGTWSDTSSGGPARAYPVGGLCYYLSPPESFGSVLEDPVHAVVYIVFMLGSCAFFSKTWIEVSGSSAKDVAKQLKEQQMV MRGHRETSMVHELNRYIPTAAAFGGLCIGALSVLADFLGAIGSGTGILLAVTIIYQYFEIFVKEQSEVGSMGALLF P61619 Protein transport protein Sec61 subunit beta Homo sapiens 0.009987456 1 LEVO MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAGRTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLF IASVFMLHIWGKYTRS P60468 Protein transport protein Sec61 subunit gamma Homo sapiens 0.007752324999999999 1 LEVO MDQVMQFVEPSRQFVKDSIRLVKRCTKPDRKEFQKIAMATAIGFAIMGFIGFFVKLIHIPINNIIVGG P60059 Stress-associated endoplasmic reticulum protein 1 Homo sapiens 0.007384762 1 LEVO MVAKQRIRMANEKHSKNITQRGNVAKTSRNAPEEKASVGPWLLALFIFVVCGSAIFQIIQSIRMGM Q9Y6X1 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A Homo sapiens 0.08060732000000001 1 LEVO MTKFGFLRLSYEKQDTLLKLLILSMAAVLSFSTRLFAVLRFESVIHEFDPYFNYRTTRFLAEEGFYKFHNWFDDRAWYPL GRIIGGTIYPGLMITSAAIYHVLHFFHITIDIRNVCVFLAPLFSSFTTIVTYHLTKELKDAGAGLLAAAMIAVVPGYISR SVAGSYDNEGIAIFCMLLTYYMWIKAVKTGSICWAAKCALAYFYMVSSWGGYVFLINLIPLHVLVLMLTGRFSHRIYVAY CTVYCLGTILSMQISFVGFQPVLSSEHMAAFGVFGLCQIHAFVDYLRSKLNPQQFEVLFRSVISLVGFVLLTVGALLMLT GKISPWTGRFYSLLDPSYAKNNIPIIASVSEHQPTTWSSYYFDLQLLVFMFPVGLYYCFSNLSDARIFIIMYGVTSMYFS AVMVRLMLVLAPVMCILSGIGVSQVLSTYMKNLDISRPDKKSKKQQDSTYPIKNEVASGMILVMAFFLITYTFHSTWVTS EAYSSPSIVLSARGGDGSRIIFDDFREAYYWLRHNTPEDAKVMSWWDYGYQITAMANRTILVDNNTWNNTHISRVGQAMA STEEKAYEIMRELDVSYVLVIFGGLTGYSSDDINKFLWMVRIGGSTDTGKHIKENDYYTPTGEFRVDREGSPVLLNCLMY KMCYYRFGQVYTEAKRPPGFDRVRNAEIGNKDFELDVLEEAYTTEHWLVRIYKVKDLDNRGLSRT P46977 2.4.99.18 Oligosaccharyltransferase complex subunit OSTC Homo sapiens 0.016844215 1 LEVO METLYRVPFLVLECPNLKLKKPPWLHMPSAMTVYALVVVSYFLITGGIIYDVIVEPPSVGSMTDEHGHQRPVAFLAYRVN GQYIMEGLASSFLFTMGGLGFIILDRSNAPNIPKLNRFLLLFIGFVCVLLSFFMARVFMRMKLPGYLMG Q9NRP0 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4 Homo sapiens 0.004196004 1 LEVO MITDVQLAIFANMLGVSLFLLVVLYHYVAVNNPKKQE P0C6T2 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit TMEM258 Homo sapiens 0.009083804 1 LEVO MELEAMSRYTSPVNPAVFPHLTVVLLAIGMFFTAWFFVYEVTSTKYTRDIYKELLISLVASLFMGFGVLFLLLWVGIYV P61165 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1 Homo sapiens 0.012503631 1 LEVO MSASVVSVISRFLEEYLSSTPQRLKLLDAYLLYILLTGALQFGYCLLVGTFPFNSFLSGFISCVGSFILAVCLRIQINPQ NKADFQGISPERAFADFLFASTILHLVVMNFVG P61803 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit KCP2 Homo sapiens 0.014689456 1 LEVO MVVGTGTSLALSSLLSLLLFAGMQMYSRQLASTEWLTIQGGLLGSGLFVFSLTAFNNLENLVFGKGFQAKIFPEILLCLL LALFASGLIHRVCVTTCFIFSMVGLYYINKISSTLYQAAAPVLTPAKVTGKSKKRN Q8N6L1 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit Homo sapiens 0.050854578 1 LEVO MGYFRCARAGSFGRRRKMEPSTAARAWALFWLLLPLLGAVCASGPRTLVLLDNLNVRETHSLFFRSLKDRGFELTFKTAD DPSLSLIKYGEFLYDNLIIFSPSVEDFGGNINVETISAFIDGGGSVLVAASSDIGDPLRELGSECGIEFDEEKTAVIDHH NYDISDLGQHTLIVADTENLLKAPTIVGKSSLNPILFRGVGMVADPDNPLVLDILTGSSTSYSFFPDKPITQYPHAVGKN TLLIAGLQARNNARVIFSGSLDFFSDSFFNSAVQKAAPGSQRYSQTGNYELAVALSRWVFKEEGVLRVGPVSHHRVGETA PPNAYTVTDLVEYSIVIQQLSNGKWVPFDGDDIQLEFVRIDPFVRTFLKKKGGKYSVQFKLPDVYGVFQFKVDYNRLGYT HLYSSTQVSVRPLQHTQYERFIPSAYPYYASAFSMMLGLFIFSIVFLHMKEKEKSD P39656 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 Homo sapiens 0.068656156 1 LEVO MEAPAAGLFLLLLLGTWAPAPGSASSEAPPLINEDVKRTVDLSSHLAKVTAEVVLAHLGGGSTSRATSFLLALEPELEAR LAHLGVQVKGEDEEENNLEVRETKIKGKSGRFFTVKLPVALDPGAKISVIVETVYTHVLHPYPTQITQSEKQFVVFEGNH YFYSPYPTKTQTMRVKLASRNVESYTKLGNPTRSEDLLDYGPFRDVPAYSQDTFKVHYENNSPFLTITSMTRVIEVSHWG NIAVEENVDLKHTGAVLKGPFSRYDYQRQPDSGISSIRSFKTILPAAAQDVYYRDEIGNVSTSHLLILDDSVEMEIRPRF PLFGGWKTHYIVGYNLPSYEYLYNLGDQYALKMRFVDHVFDEQVIDSLTVKIILPEGAKNIEIDSPYEISRAPDELHYTY LDTFGRPVIVAYKKNLVEQHIQDIVVHYTFNKVLMLQEPLLVVAAFYILFFTVIIYVRLDFSITKDPAAEARMKVACITE QVLTLVNKRIGLYRHFDETVNRYKQSRDISTLNSGKKSLETEHKALTSEIALLQSRLKTEGSDLCDRVSEMQKLDAQVKE LVLKSAVEAERLVAGKLKKDTYIENEKLISGKRQELVTKIDHILDAL P04843 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 Homo sapiens 0.069347508 1 LEVO MAPPGSSTVFLLALTIIASTWALTPTHYLTKHDVERLKASLDRPFTNLESAFYSIVGLSSLGAQVPDAKKACTYIRSNLD PSNVDSLFYAAQASQALSGCEISISNETKDLLLAAVSEDSSVTQIYHAVAALSGFGLPLASQEALSALTARLSKEETVLA TVQALQTASHLSQQADLRSIVEEIEDLVARLDELGGVYLQFEEGLETTALFVAATYKLMDHVGTEPSIKEDQVIQLMNAI FSKKNFESLSEAFSVASAAAVLSHNRYHVPVVVVPEGSASDTHEQAILRLQVTNVLSQPLTQATVKLEHAKSVASRATVL QKTSFTPVGDVFELNFMNVKFSSGYYDFLVEVEGDNRYIANTVELRVKISTEVGITNVDLSTVDKDQSIAPKTTRVTYPA KAKGTFIADSHQNFALFFQLVDVNTGAELTPHQTFVRLHNQKTGQEVVFVAEPDNKNVYKFELDTSERKIEFDSASGTYT LYLIIGDATLKNPILWNVADVVIKFPEEEAPSTVLSQNLFTPKQEIQHLFREPEKRPPTVVSNTFTALILSPLLLLFALW IRIGANVSNFTFAPSTIIFHLGHAAMLGLMYVYWTQLNMFQTLKYLAILGSVTFLAGNRMLAQQAVKRTAH P04844 Endoplasmin Homo sapiens 0.096254234 1 LEVO MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQA EVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELV KNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNT LGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKK PKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDE YGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKY NDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLK KGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRL TESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFET ATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAEK DEL(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) P14625 3.6.4.- phosphono [(3~{R},6~{E},10~{E})-3,7,11,15-tetramethylhexadeca-6,10,14-trienyl] hydrogen phosphate 0.000452459 1 ELU singleParticle particle 0.056 7.4 50.0 C8H18N2O4S HEPES, pH 7.4 200.0 CH3CO2K Potassium Acetate 2.0 MgCl2 Magnesium Chloride 1.0 C10H17N3O6S Glutathione 0.15 C56H92O29 Digitonin 0.5 C120H169N31O49S 3xFlag Peptide

HEPES buffer was adjusted to pH 7.4 with potassium hydroxide.

Quantifoil R1.2/1.3 GOLD 400 CARBON CONTINUOUS 2.0 GLOW DISCHARGE 25 ETHANE 100 295 FEI VITROBOT MARK IV

Three microliters of affinity-purified RTCs were applied to the grid, incubated for 60 seconds, blotted for 7 seconds, drained for 0.5 seconds, and frozen in liquid nitrogen-cooled ethane..

Native ribosome-translocon complexes isolated from HEK293 microsomes by affinity purification via a Flag-tag on FKBP11

TFS KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 2.7 0.9 1.9000000000000001 53000.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN GATAN K3 BIOQUANTUM (6k x 4k) 1 6569 60.0 1 741983 NONE

Unpublished ribosome-translocon map

1 4.1 FSC 0.143 CUT-OFF RELION 5.0 55750 MAXIMUM LIKELIHOOD MAXIMUM LIKELIHOOD 4 emd_72945.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 416 416 416 0 0 0 416 416 416 349.44 349.44 349.44 90.0 90.0 90.0 X Y Z -0.005121462 0.01718846 0.00082456216 0.00093151646 0.84000003 0.84000003 0.84000003 0.00395 AUTHOR ::::EMDATABANK.org::::EMD-72945:::: Translocon-only map FLEXIBLE FIT

Tightly restrained real-space refinement in Coot and Phenix

REAL emd_72945_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 416 416 416 0 0 0 416 416 416 349.44 349.44 349.44 90.0 90.0 90.0 X Y Z -0.027325079 0.035367746 0.00082867633 0.002398174 0.84000003 0.84000003 0.84000003 AUTHOR ::::EMDATABANK.org::::EMD-72945:::: half-map 1 emd_72945_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 416 416 416 0 0 0 416 416 416 349.44 349.44 349.44 90.0 90.0 90.0 X Y Z -0.025404448 0.0349382 0.00082051853 0.0024280625 0.84000003 0.84000003 0.84000003 AUTHOR ::::EMDATABANK.org::::EMD-72945:::: half-map 2