EMDB-14243: cryoEM structure of human Nup155 (residues 19-981)

Basic information

Entry

Database: EMDB / ID: EMD-14243

• Title: cryoEM structure of human Nup155 (residues 19-981)

Sample

• Organelle or cellular component: Nup155 from homo sapiens
  • Protein or peptide: Nuclear pore complex protein Nup155

• Keywords: nucleoporin / beta-propeller / STRUCTURAL PROTEIN

Function / homology

Function:

nuclear pore inner ring / protein localization to nuclear inner membrane / nuclear envelope organization / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / atrial cardiac muscle cell action potential / Nuclear Pore Complex (NPC) Disassembly / Transport of Ribonucleoproteins into the Host Nucleus / Regulation of Glucokinase by Glucokinase Regulatory Protein / Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC) / miRNA processing / Transport of the SLBP independent Mature mRNA / Transport of the SLBP Dependant Mature mRNA / NS1 Mediated Effects on Host Pathways / SUMOylation of SUMOylation proteins / Transport of Mature mRNA Derived from an Intronless Transcript / structural constituent of nuclear pore / Rev-mediated nuclear export of HIV RNA / SUMOylation of RNA binding proteins / Nuclear import of Rev protein / RNA export from nucleus / NEP/NS2 Interacts with the Cellular Export Machinery / tRNA processing in the nucleus / Transport of Mature mRNA derived from an Intron-Containing Transcript / Postmitotic nuclear pore complex (NPC) reformation / nucleocytoplasmic transport / Viral Messenger RNA Synthesis / SUMOylation of ubiquitinylation proteins / Vpr-mediated nuclear import of PICs / SUMOylation of DNA replication proteins / Regulation of HSF1-mediated heat shock response / mRNA export from nucleus / nuclear pore / SUMOylation of DNA damage response and repair proteins / SUMOylation of chromatin organization proteins / HCMV Late Events / Transcriptional regulation by small RNAs / ISG15 antiviral mechanism / HCMV Early Events / protein import into nucleus / nuclear envelope / snRNP Assembly / nuclear membrane / SARS-CoV-2 activates/modulates innate and adaptive immune responses / membrane / cytosol

Domain/homology:

Nucleoporin, Nup155-like, C-terminal, subdomain 3 / Nucleoporin, Nup155-like / Nucleoporin, Nup155-like, C-terminal, subdomain 1 / Nucleoporin, Nup155-like, C-terminal, subdomain 2 / Nucleoporin, Nup133/Nup155-like, C-terminal / Non-repetitive/WGA-negative nucleoporin C-terminal / Nucleoporin, Nup133/Nup155-like, N-terminal / Nup133 N terminal like

Component:

Nuclear pore complex protein Nup155

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.0 Å
• Authors: Taniguchi R / Beck M

Funding support

Germany, 1 items

Organization	Grant number	Country
Max Planck Society		Germany

• Citation: Journal: Science / Year: 2022; Title: AI-based structure prediction empowers integrative structural analysis of human nuclear pores.; Authors: Shyamal Mosalaganti / Agnieszka Obarska-Kosinska / Marc Siggel / Reiya Taniguchi / Beata Turoňová / Christian E Zimmerli / Katarzyna Buczak / Florian H Schmidt / Erica Margiotta / Marie-Therese Mackmull / Wim J H Hagen / Gerhard Hummer / Jan Kosinski / Martin Beck / ; Abstract: INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of ∼120 MDa, the human NPC is one of the larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf-fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma-tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal-lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra-tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc-tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf-fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight-forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol-ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)-based prediction to generate an exten-sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac-terized. Benchmarking against previous and unpublished x-ray and cryo-electron micros-copy structures revealed unprecedented accu-racy. We obtained well-resolved cryo-electron tomographic maps of both the constricted and dilated conformational states of the hu-man NPC. Using integrative modeling, we fit-ted the structural models of individual NUPs into the cryo-electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf-fold. We elucidated in great detail how mem-brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en-vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re-solved model covers >90% of the human NPC scaffold. It captures conforma-tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy-namic model of the NPC. Our study exempli-fies how AI-based structure prediction may accelerate the elucidation of subcellular ar-chitecture at atomic resolution. [Figure: see text].

History

Deposition	Feb 3, 2022	-
Header (metadata) release	Jun 8, 2022	-
Map release	Jun 8, 2022	-
Update	Jul 17, 2024	-
Current status	Jul 17, 2024	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_14243.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 0.9344 Å

Density

Contour Level	By AUTHOR: 0.0085
Minimum - Maximum	-0.027457202 - 0.036329564
Average (Standard dev.)	0.00008757497 (±0.0009964276)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 200 200 200 Spacing 200 200 200
Cell	A=B=C: 186.87999 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_14243_msk_1.map

Half map: #1

• File: emd_14243_half_map_1.map

Half map: #2

• File: emd_14243_half_map_2.map

Sample components

Entire : Nup155 from homo sapiens

• Entire: Name: Nup155 from homo sapiens

Components

• Organelle or cellular component: Nup155 from homo sapiens
  • Protein or peptide: Nuclear pore complex protein Nup155

Supramolecule #1: Nup155 from homo sapiens

• Supramolecule: Name: Nup155 from homo sapiens / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: Nuclear pore complex protein Nup155

• Macromolecule: Name: Nuclear pore complex protein Nup155 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 154.999672 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

String:

GPPSSLLGAA MPASTSAAAL QEALENAGRL IDRQLQEDRM YPDLSELLMV SAPNNPTVSG MSDMDYPLQG PGLLSVPNLP EISSIRRVP LPPELVEQFG HMQCNCMMGV FPPISRAWLT IDSDIFMWNY EDGGDLAYFD GLSETILAVG LVKPKAGIFQ P HVRHLLVL ATPVDIVILG LSYANLQTGS GVLNDSLSGG MQLLPDPLYS LPTDNTYLLT ITSTDNGRIF LAGKDGCLYE VA YQAEAGW FSQRCRKINH SKSSEDDPIL QIAIDNSRNI LYTRSEKGVI QVYDLGQDGQ GMSRVASVSQ NAIVSAAGNI ART IDRSVF KPIVQIAVIE NSESLDCQLL AVTHAGVRLY FSTCPFRQPL ARPNTLTLVH VRLPPGFSAS STVEKPSKVH RALY SKGIL LMAASENEDN DILWCVNHDT FPFQKPMMET QMTAGVDGHS WALSAIDELK VDKIITPLNK DHIPITDSPV VVQQH MLPP KKFVLLSAQG SLMFHKLRPV DQLRHLLVSN VGGDGEEIER FFKLHQEDQA CATCLILACS TAACDREVSA WATRAF FRY GGEAQMRFPT TLPPPSNVGP ILGSPVYSSS PVPSGSPYPN PSFLGTPSHG IQPPAMSTPV CALGNPATQA TNMSCVT GP EIVYSGKHNG ICIYFSRIMG NIWDASLVVE RIFKSGNREI TAIESSVPCQ LLESVLQELK GLQEFLDRNS QFAGGPLG N PNTTAKVQQR LIGFMRPENG NPQQMQQELQ RKFHEAQLSE KISLQAIQQL VRKSYQALAL WKLLCEHQFT IIVAELQKE LQEQLKITTF KDLVIRDKEL TGALIASLIN CYIRDNAAVD GISLHLQDIC PLLYSTDDAI CSKANELLQR SRQVQNKTEK ERMLRESLK EYQKISNQVD LSNVCAQYRQ VRFYEGVVEL SLTAAEKKDP QGLGLHFYKH GEPEEDIVGL QAFQERLNSY K CITDTLQE LVNQSKAAPQ SPSVPKKPGP PVLSSDPNML SNEEAGHHFE QMLKLSQRSK DELFSIALYN WLIQVDLADK LL QVASPFL EPHLVRMAKV DQNRVRYMDL LWRYYEKNRS FSNAARVLSR LADMHSTEIS LQQRLEYIAR AILSAKSSTA ISS IAADGE FLHELEEKME VARIQLQIQE TLQRQYSHHS SVQDAVSQLD SELMDITKLY GEFADPFKLA ECKLAIIHCA GYSD PILVQ TLWQDIIEKE LSDSVTLSSS DRMHALSLKI VLLGKIYAGT PRFFPLDFIV QFLEQQVCTL NWDVGFVIQT MNEIG VPLP RLLEVYDQLF KSRDPFWNRM KKPLHLLDCI HVLLIRYVEN PSQVLNCERR RFTNLCLDAV CGYLVELQSM SSSVAV QAI TGNFKSLQAK LERLHSAWSH PQFEK

UniProtKB: Nuclear pore complex protein Nup155

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.4 mg/mL
• Buffer: pH: 7.5
• Grid: Model: UltrAuFoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
• Image recording: Film or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 5901 / Average exposure time: 5.85 sec. / Average electron dose: 50.0 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

Startup model

Type of model: PDB ENTRY
PDB model - PDB ID:

5hax

• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 98742
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)

Atomic model buiding 1

Initial model

PDB ID:

5hax

Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model

• Refinement: Space: REAL / Protocol: AB INITIO MODEL

Output model

PDB-7r1y:
cryoEM structure of human Nup155 (residues 19-981)