EMDB-25687: CryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in...

Basic information

Entry

Database: EMDB / ID: EMD-25687

• Title: CryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in complex with NRP2 and neutralizing fabs 8I21 and 13H11
• Map data: Composite map of combined focused refinement maps. Used for model refinements.

Sample

• Complex: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A bound to Neuropilin 2 and fabs of human neutralizing antibodies 8I21 and 13H11
  • Protein or peptide: x 10 types
• Ligand: x 2 types

Function / homology

Function:

vestibulocochlear nerve structural organization / dorsal root ganglion morphogenesis / ventral trunk neural crest cell migration / sympathetic neuron projection guidance / facioacoustic ganglion development / trigeminal ganglion development / trigeminal nerve structural organization / sensory neuron axon guidance / facial nerve structural organization / gonadotrophin-releasing hormone neuronal migration to the hypothalamus / branchiomotor neuron axon guidance / axon extension involved in axon guidance / NrCAM interactions / sympathetic neuron projection extension / Neurophilin interactions with VEGF and VEGFR / sympathetic ganglion development / neural crest cell migration involved in autonomic nervous system development / vascular endothelial growth factor receptor activity / nerve development / semaphorin receptor complex / outflow tract septum morphogenesis / semaphorin receptor activity / regulation of postsynapse organization / negative chemotaxis / cytokine binding / growth factor binding / semaphorin-plexin signaling pathway / positive regulation of endothelial cell proliferation / cellular response to leukemia inhibitory factor / host cell endosome membrane / positive regulation of endothelial cell migration / axon guidance / signaling receptor activity / heparin binding / host cell Golgi apparatus / postsynaptic membrane / angiogenesis / entry receptor-mediated virion attachment to host cell / cell adhesion / symbiont entry into host cell / axon / fusion of virus membrane with host plasma membrane / viral envelope / glutamatergic synapse / host cell plasma membrane / virion membrane / extracellular region / membrane / identical protein binding / metal ion binding / plasma membrane

Domain/homology:

Herpesvirus UL130, cytomegalovirus / HCMV glycoprotein pUL130 / Cytomegalovirus glycoprotein L / Cytomegalovirus glycoprotein L / Herpesvirus glycoprotein H main domain / Herpesvirus glycoprotein H / Herpesvirus glycoprotein H, C-terminal / Herpesvirus glycoprotein H, C-terminal domain superfamily / Herpesvirus glycoprotein H C-terminal domain / Neuropilin / Neuropilin, C-terminal / C-terminal domain of neuropilin glycoprotein / Domain in meprin, A5, receptor protein tyrosine phosphatase mu (and others) / MAM domain, meprin/A5/mu / MAM domain / Coagulation factors 5/8 type C domain (FA58C) signature 2. / MAM domain profile. / CUB domain / Domain first found in C1r, C1s, uEGF, and bone morphogenetic protein. / CUB domain / CUB domain profile. / Coagulation factors 5/8 type C domain (FA58C) signature 1. / Spermadhesin, CUB domain superfamily / Coagulation factor 5/8 C-terminal domain, discoidin domain / Coagulation factors 5/8 type C domain (FA58C) profile. / F5/8 type C domain / Coagulation factor 5/8 C-terminal domain / Galactose-binding-like domain superfamily / Concanavalin A-like lectin/glucanase domain superfamily

Component:

Envelope glycoprotein H / Neuropilin-2 / UL128 / UL130 / Envelope glycoprotein L / UL131A

• Biological species: Human betaherpesvirus 5 / Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.1 Å
• Authors: Kschonsak M / Johnson MC / Schelling R / Green EM / Rouge L / Ho H / Patel N / Kilic C / Kraft E / Arthur CP / Rohou AL / Comps-Agrar L / Martinez-Martin N / Perez L / Payandeh J / Ciferri C

Funding support

1 items

Organization	Grant number	Country
Not funded

• Citation: Journal: Sci Adv / Year: 2022; Title: Structural basis for HCMV Pentamer receptor recognition and antibody neutralization.; Authors: Marc Kschonsak / Matthew C Johnson / Rachel Schelling / Evan M Green / Lionel Rougé / Hoangdung Ho / Nidhi Patel / Cem Kilic / Edward Kraft / Christopher P Arthur / Alexis L Rohou / Laetitia Comps-Agrar / Nadia Martinez-Martin / Laurent Perez / Jian Payandeh / Claudio Ciferri / ; Abstract: Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.

History

Deposition	Dec 10, 2021	-
Header (metadata) release	Mar 23, 2022	-
Map release	Mar 23, 2022	-
Update	Mar 23, 2022	-
Current status	Mar 23, 2022	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_25687.map.gz / Format: CCP4 / Size: 87.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Composite map of combined focused refinement maps. Used for model refinements.
• Voxel size: X=Y=Z: 1.3514 Å

Density

Contour Level	By AUTHOR: 0.8
Minimum - Maximum	0.0 - 15.305042
Average (Standard dev.)	0.011590637 (±0.18164654)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 284 284 284 Spacing 284 284 284
Cell	A=B=C: 383.7976 Å α=β=γ: 90.0 °

Supplemental data

Additional map: Non-sharpened, full map of overall (non-focused)refinement

• File: emd_25687_additional_1.map
• Annotation: Non-sharpened, full map of overall (non-focused)refinement

Additional map: Non-sharpened, full map of focused refinement on 8I21-UL128 region

• File: emd_25687_additional_10.map
• Annotation: Non-sharpened, full map of focused refinement on 8I21-UL128 region

Additional map: Half map 1 of focused refinement on 8I21-UL128 region

• File: emd_25687_additional_11.map
• Annotation: Half map 1 of focused refinement on 8I21-UL128 region

Additional map: Density modified map of focused refined 8I12-UL128 region....

• File: emd_25687_additional_12.map
• Annotation: Density modified map of focused refined 8I12-UL128 region. Map used for composite map generation.

Additional map: Density modified map of focused refined NRP2-UL region....

• File: emd_25687_additional_13.map
• Annotation: Density modified map of focused refined NRP2-UL region. Map used for composite map generation.

Additional map: Non-sharpened, full map of focused refinement on NRP2-UL region

• File: emd_25687_additional_2.map
• Annotation: Non-sharpened, full map of focused refinement on NRP2-UL region

Additional map: Half map 1 of focused refinement on NRP2-UL region

• File: emd_25687_additional_3.map
• Annotation: Half map 1 of focused refinement on NRP2-UL region

Additional map: Half map 2 of focused refinement on NRP2-UL region

• File: emd_25687_additional_4.map
• Annotation: Half map 2 of focused refinement on NRP2-UL region

Additional map: Half map 2 of focused refinement on 8I21-UL128 region

• File: emd_25687_additional_5.map
• Annotation: Half map 2 of focused refinement on 8I21-UL128 region

Additional map: Non-sharpened, full map of focused refinement on 13H11-gH region

• File: emd_25687_additional_6.map
• Annotation: Non-sharpened, full map of focused refinement on 13H11-gH region

Additional map: Half map 1 of focused refinement on 13H11-gH region

• File: emd_25687_additional_7.map
• Annotation: Half map 1 of focused refinement on 13H11-gH region

Additional map: Half map 2 of focused refinement on 13H11-gH region

• File: emd_25687_additional_8.map
• Annotation: Half map 2 of focused refinement on 13H11-gH region

Additional map: Density modified map of focused refined 13H11-gH region....

• File: emd_25687_additional_9.map
• Annotation: Density modified map of focused refined 13H11-gH region. Map used for composite map generation.

Sample components

Entire : Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A ...

• Entire: Name: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A bound to Neuropilin 2 and fabs of human neutralizing antibodies 8I21 and 13H11

Components

• Complex: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A bound to Neuropilin 2 and fabs of human neutralizing antibodies 8I21 and 13H11
  • Protein or peptide: Envelope glycoprotein H
  • Protein or peptide: Envelope glycoprotein L
  • Protein or peptide: Envelope protein UL128
  • Protein or peptide: Envelope glycoprotein UL130
  • Protein or peptide: Envelope protein UL131A
  • Protein or peptide: Neuropilin-2Neuropilin
  • Protein or peptide: Fab 8I21 heavy chain
  • Protein or peptide: Fab 13H11 heavy chain
  • Protein or peptide: Fab 13H11 light chain
  • Protein or peptide: Fab 8I21 light chain
• Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
• Ligand: CALCIUM IONCalcium

Supramolecule #1: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A ...

• Supramolecule: Name: Pentameric complex of HCMV proteins gH, gL, UL128, UL130, UL131A bound to Neuropilin 2 and fabs of human neutralizing antibodies 8I21 and 13H11; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
• Molecular weight: Theoretical: 360 KDa

Macromolecule #1: Envelope glycoprotein H

• Macromolecule: Name: Envelope glycoprotein H / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human betaherpesvirus 5
• Molecular weight: Theoretical: 87.311273 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MRPGLPSYLI ILAVCLFSHL LSSRYGAEAV SEPLDKAFHL LLNTYGRPIR FLRENTTQCT YNSSLRNSTV VRENAISFNF FQSYNQYYV FHMPRCLFAG PLAEQFLNQV DLTETLERYQ QRLNTYALVS KDLASYRSFS QQLKAQDSLG EQPTTVPPPI D LSIPHVWM PPQTTPHGWT ESHTTSGLHR PHFNQTCILF DGHDLLFSTV TPCLHQGFYL IDELRYVKIT LTEDFFVVTV SI DDDTPML LIFGHLPRVL FKAPYQRDNF ILRQTEKHEL LVLVKKDQLN RHSYLKDPDF LDAALDFNYL DLSALLRNSF HRY AVDVLK SGRCQMLDRR TVEMAFAYAL ALFAAARQEE AGAQVSVPRA LDRQAALLQI QEFMITCLSQ TPPRTTLLLY PTAV DLAKR ALWTPNQITD ITSLVRLVYI LSKQNQQHLI PQWALRQIAD FALKLHKTHL ASFLSAFARQ ELYLMGSLVH SMLVH TTER REIFIVETGL CSLAELSHFT QLLAHPHHEY LSDLYTPCSS SGRRDHSLER LTRLFPDATV PATVPAALSI LSTMQP STL ETFPDLFCLP LGESFSALTV SEHVSYIVTN QYLIKGISYP VSTTVVGQSL IITQTDSQTK CELTRNMHTT HSITVAL NI SLENCAFCQS ALLEYDDTQG VINIMYMHDS DDVLFALDPY NEVVVSSPRT HYLMLLKNGT VLEVTDVVVD ATDGTKLG P EQKLISEEDL NSAVDGSGLN DIFEAQKIEW HENLYFQGHH HHHHHH

Macromolecule #2: Envelope glycoprotein L

• Macromolecule: Name: Envelope glycoprotein L / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human betaherpesvirus 5
• Molecular weight: Theoretical: 30.846492 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MCRRPDCGFS FSPGPVILLW CCLLLPIVSS AAVSVAPTAA EKVPAECPEL TRRCLLGEVF EGDKYESWLR PLVNVTGRDG PLSQLIRYR PVTPEAANSV LLDEAFLDTL ALLYNNPDQL RALLTLLSSD TAPRWMTVMR GYSECGDGSP AVYTCVDDLC R GYDLTRLS YGRSIFTEHV LGFELVPPSL FNVVVAIRNE ATRTNRAVRL PVSTAAAPEG ITLFYGLYNA VKEFCLRHQL DP PLLRHLD KYYAGLPPEL KQTRVNLPAH SRYGPQAVDA R

Macromolecule #3: Envelope protein UL128

• Macromolecule: Name: Envelope protein UL128 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human betaherpesvirus 5
• Molecular weight: Theoretical: 19.746023 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MSPKNLTPFL TALWLLLGHS RVPRVRAEEC CEFINVNHPP ERCYDFKMCN RFTVALRCPD GEVCYSPEKT AEIRGIVTTM THSLTRQVV HNKLTSCNYN PLYLEADGRI RCGKVNDKAQ YLLGAAGSVP YRWINLEYDK ITRIVGLDQY LESVKKHKRL D VCRAKMGY MLQ

Macromolecule #4: Envelope glycoprotein UL130

• Macromolecule: Name: Envelope glycoprotein UL130 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human betaherpesvirus 5
• Molecular weight: Theoretical: 28.866811 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MLRLLLRHHF HCLLLCAVWA TPCLASPWFT LTANQNPSPP WSKLTYPKPH DAATFYCPFL YPSPPRSPSQ FSGFQRVSTG PECRNETLY LLYNREGQTL VERSSTWVKK VIWYLSGRNQ TILQRMPRTA SKPSDGNVQI SVEDAKIFGA HMVPKQTKLL R FVVNDGTR YQMCVMKLES WAHVFRDYSV SFQVRLTFTE ANNQTYTFCT HPNLIVGSEN LYFQGSAWSH PQFEKGGGSG GG SGGGSAW SHPQFEK

Macromolecule #5: Envelope protein UL131A

• Macromolecule: Name: Envelope protein UL131A / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human betaherpesvirus 5
• Molecular weight: Theoretical: 15.011043 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MRLCRVWLSV CLCAVVLGQC QRETAEKNDY YRVPHYWDAC SRALPDQTRY KYVEQLVDLT LNYHYDASHG LDNFDVLKRI NVTEVSLLI SDFRRQNRRG GTNKRTTFNA AGSLAPHARS LEFSVRLFAN

Macromolecule #6: Neuropilin-2

• Macromolecule: Name: Neuropilin-2 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 98.923211 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MDMFPLTWVF LALYFSRHQV RGQPDPPCGG RLNSKDAGYI TSPGYPQDYP SHQNCEWIVY APEPNQKIVL NFNPHFEIEK HDCKYDFIE IRDGDSESAD LLGKHCGNIA PPTIISSGSM LYIKFTSDYA RQGAGFSLRY EIFKTGSEDC SKNFTSPNGT I ESPGFPEK YPHNLDCTFT ILAKPKMEII LQFLIFDLEH DPLQVGEGDC KYDWLDIWDG IPHVGPLIGK YCGTKTPSEL RS STGILSL TFHTDMAVAK DGFSARYYLV HQEPLENFQC NVPLGMESGR IANEQISASS TYSDGRWTPQ QSRLHGDDNG WTP NLDSNK EYLQVDLRFL TMLTAIATQG AISRETQNGY YVKSYKLEVS TNGEDWMVYR HGKNHKVFQA NNDATEVVLN KLHA PLLTR FVRIRPQTWH SGIALRLELF GCRVTDAPCS NMLGMLSGLI ADSQISASST QEYLWSPSAA RLVSSRSGWF PRIPQ AQPG EEWLQVDLGT PKTVKGVIIQ GARGGDSITA VEARAFVRKF KVSYSLNGKD WEYIQDPRTQ QPKLFEGNMH YDTPDI RRF DPIPAQYVRV YPERWSPAGI GMRLEVLGCD WTDSKPTVET LGPTVKSEET TTPYPTEEEA TECGENCSFE DDKDLQL PS GFNCNFDFLE EPCGWMYDHA KWLRTTWASS SSPNDRTFPD DRNFLRLQSD SQREGQYARL ISPPVHLPRS PVCMEFQY Q ATGGRGVALQ VVREASQESK LLWVIREDQG GEWKHGRIIL PSYDMEYQIV FEGVIGKGRS GEIAIDDIRI STDVPLENC MEPISAFAGE NFKVDIPEIH EREGYEDEID DEYEVDWSNS SSATSGSGAP STDKEKSWLY TLDPGNSDYK DDDDK

Macromolecule #7: Fab 8I21 heavy chain

• Macromolecule: Name: Fab 8I21 heavy chain / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 26.816311 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYALVELVES GGGVVQPGRS LRLSCAASGF TFSSDGMHWV RQSPGRGLEW VAFISSDGST PYYADSVKG RFTISRDNSK NTLYLQMNSL RAEDTAMYFC AKDWALFRWL RTFDHWGQGT LVTVSSASTK GPSVFPLAPS S KSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VD KKVEPKS CD

Macromolecule #8: Fab 13H11 heavy chain

• Macromolecule: Name: Fab 13H11 heavy chain / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 26.600086 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYAQVQLVQS GAEVKKPGAS VKVSCKASGY TFTNYYIHWV RQAPGQGLEW MGIIHPSSGG TSYAQKFQG RVTMTRDTST STVSMDLSSL RSEDTAVYYC GRAFRILGLS DVFVNDWGQG TVVTVSSAST KGPSVFPLAP S SKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KV DKKVEPK SCD

Macromolecule #9: Fab 13H11 light chain

• Macromolecule: Name: Fab 13H11 light chain / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 25.78002 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYADIQMTQS PSSLSASVGD RVTITCRASQ GINNYLAWYQ QKPGKVPKLL IYAASTLQSG VPSRFSGSG SGTAFTLTIL SLQPEDVATY YCQKYNSAPF TFGPGTKVDI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL N NFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

Macromolecule #10: Fab 8I21 light chain

• Macromolecule: Name: Fab 8I21 light chain / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 25.99508 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKKNIAFLLA SMFVFSIATN AYAETVMTQS PATLSVSPGG RATLSCRASQ SVGINLAWYQ QKPGQAPRLL IYGASTRASG FPARFSGSG SGTEFTLTIT SLQSEDFAVY YCQQYNDWPP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL L NNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

Macromolecule #11: 2-acetamido-2-deoxy-beta-D-glucopyranose

• Macromolecule: Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 11 / Number of copies: 9 / Formula: NAG
• Molecular weight: Theoretical: 221.208 Da

Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

Macromolecule #12: CALCIUM ION

• Macromolecule: Name: CALCIUM ION / type: ligand / ID: 12 / Number of copies: 1 / Formula: CA
• Molecular weight: Theoretical: 40.078 Da

Experimental details

Structure determination

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

Sample preparation

• Concentration: 1 mg/mL

Buffer

pH: 7.5
Component:

Concentration	Formula	Name
300.0 mM	NaClSodium chloride	sodium chloride
25.0 mM	HEPES	HEPES

• Grid: Model: UltrAuFoil R0.6/1 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP / Details: blot for 3.5 seconds before plunging.
• Details: Sample was mildly crosslinked with 0.025% glutaraldehyde, incubated for 10 min at room temperature and quenched with 9 mM TRIS pH 7.5

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 165000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 4 / Number real images: 21357 / Average exposure time: 10.0 sec. / Average electron dose: 52.0 e/Ų; Details: Images were collected in movie-mode at 4 frames/second.
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 2532206 / Details: template-matching particle picking
• CTF correction: Software - Name: CTFFIND (ver. 4.1.13)
• Startup model: Type of model: OTHER; Details: ab-initio 3D model generation from select particles within cisTEM
• Initial angle assignment: Type: OTHER / Software - Name: cisTEM (ver. 1.02) / Details: Auto-refine in cisTEM
• Final 3D classification: Number classes: 100 / Software - Name: RELION (ver. 3.1)
• Final angle assignment: Type: OTHER / Software - Name: cisTEM (ver. 1.02); Details: Manual refine in cisTEM. Map was divided in 3 sections to refine map with focused refinements.
• Final reconstruction: Number classes used: 44 / Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.02); Details: A composite map was generated from the three individual focused 3D maps.; Number images used: 2252924
• Details: Movie frames were corrected for motion and aligned. Images with a CTF fit resolution of 6.0 A or better were selected for particle picking.

Atomic model buiding 1

Initial model

(PDB ID:

2qqo

,

5vob

)

Output model

PDB-7t4s:
CryoEM structure of the HCMV Pentamer gH/gL/UL128/UL130/UL131A in complex with NRP2 and neutralizing fabs 8I21 and 13H11