Protein structure prediction using Phyre2 and ... · The Phyre2 web portal for protein modeling,...
Transcript of Protein structure prediction using Phyre2 and ... · The Phyre2 web portal for protein modeling,...
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Protein structure prediction using Phyre2
and understanding genetic variantsProf Michael SternbergDr Lawrence KelleyMr Stefans Mezulis
Dr Chris Yates
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Today
• 10.00 – 11.00 Lecture• 11.00 – 11.30 Tea/Coffee• Courtyard, West Medical Building• 11.30 – 1.00 Hands on workshop using
Phyre2
• Computer Cluster 515, West Medical Building
Many thanks to Glasgow Polyomicsand Amy Cattanach
Timetable
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• Methods• Interpretation of results• Extended functionality• Proposed developments
• Publications:The Phyre2 web portal for protein modeling, prediction and analysis Kelley,LA, Mezulis S, Yates CM, Wass MN & Sternberg MJESNature Protocols 10, 845–858 (2015)
SuSPect: Enhanced Prediction of Single Amino Acid Variant (SAV) Phenotype Using Network Features. Yates CM, Filippis I, Kelley LA, Sternberg MJE. Journal of Molecular Biology.;426, 2692‐2701. (2014)
Overview
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SVYDAAAQLTADVKKDLRDSWKVIGSDKKGNGVALMTTLFADNQETIGYFKRLGNVSQGMANDKLRGHSITLMYALQNFIDQLDNPDSLDLVCS…….
Predict the 3D structure adopted by a user‐supplied protein sequence
Phyre2
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http://www.sbg.bio.ic.ac.uk/phyre2
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• “Normal” Mode• “Intensive” Mode• Advanced functions
How does Phyre2 work?
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ARDLVIPMIYCGHGY
Search the 30 million known sequences for homologues using PSI‐Blast.
Phyre2
Homologous sequences
User sequence
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ARDLVIPMIYCGHGY HMM
PSI‐Blast
Phyre2
Hidden Markov model
Capture the mutational propensities at each position in the protein
An evolutionary fingerprint
User sequence
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~ 100,000 known 3D structures
Phyre2
HAPTLVRDC…….
Extract sequence
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~ 100,000 known 3D structures
Phyre2
HAPTLVRDC…….
HMM
PSI‐Blast
Hidden Markov modelfor sequence of KNOWN structure
Extract sequence
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~ 100,000 known 3D structures
Phyre2
HMM HMM HMM
~ 100,000 hidden Markov models
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~ 100,000 known 3D structures
Phyre2
Hidden Markov Model Database of KNOWN
STRUCTURES
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ARDLVIPMIYCGHGY HMM
PSI‐Blast
Phyre2
Hidden Markov model
Capture the mutational propensities at each position in the protein
An evolutionary fingerprint
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ARDLVIPMIYCGHGY HMM
PSI‐Blast
Hidden Markov Model DB of KNOWN
STRUCTURES
Phyre2
Alignments of user sequence to known structuresranked by confidence.
ARDL--VIPMIYCGHGYAFDLCDLIPV--CGMAY
Sequence of known structure
HMM‐HMM Matching(HHsearch, Soeding)
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ARDLVIPMIYCGHGY HMM
PSI‐Blast
Hidden Markov Model DB of KNOWN
STRUCTURES
HMM‐HMM Matching(HHsearch, Soeding)
Phyre2
ARDL--VIPMIYCGHGYAFDLCDLIPV--CGMAY
Sequence of known structure
3D‐Model
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ARDLVIPMIYCGHGY HMM
PSI‐Blast
Hidden Markov Model DB of KNOWN
STRUCTURES
Phyre2
ARDL--VIPMIYCGHGYAFDLCDLIPV--CGMAY
Sequence of known structure
Very powerful –able to reliably detect extremely remote homology
Routinely creates accurate models even when sequence identity is <15%
3D‐Model
HMM‐HMM Matching(HHsearch, Soeding)
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ARDL--VIPMIYCGHGYAFDLCDLIPV--CGMAY
A
D CL
D
I
L
P CV
GY
MA
F
Query (your sequence)
Known Structure
Known 3DStructure coordinates
From alignment to crude model
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ARDL--VIPMIYCGHGYAFDLCDLIPV--CGMAY
A
D
L
I
V
P CM
GY
MA
RDel
Y
I Insertion (handled by loop modelling)
Re‐label the known structureaccording to the mapping fromthe alignment.
Homology model
Query
KnownStructure
From alignment to crude model
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Loop modelling
d
ARDAKQH
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Loop modelling
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• Insertions and deletions relative to template modelled by a loop library up to 15 aa’s in length
• Short loops (<=5) good. Longer loops less trustworthy
• Be wary of basing any interpretation of the structural effects of point mutations
Loop modelling
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Sidechain modelling
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Sidechain modelling
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Sidechain modelling
Optimisation problem
• Fit most probablerotamer at each position
• According to given backbone angles
• Whilst avoiding clashes
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• Sidechains will be modelled with ~80% accuracy IF……the backbone is correct.
• Clashes *will* sometimes occur and if frequent, indicate probably a wrong alignment or poor template
• Analyse with Phyre Investigator
Sidechain modelling
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Top model info
Secondary structure/disorder
Domain analysis
Detailed template information
Example results
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Example results
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Top model info
Secondary structure/disorder
Domain analysis
Detailed template information
Example results
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Example SS/disorder prediction
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• Based on neural networks trained on known structures.
• Given a diverse set of homologous sequences, expect ~75‐80% accuracy.
• Few or no homologous sequences? Only 60‐62% accuracy
Secondary structure and disorder
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Top model info
Secondary structure/disorder
Domain analysis
Detailed template information
Example results
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Example domain analysis
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• Local hits to different templates indicate domain structure of your protein
• Multiple domains can be linked using ‘Intensive mode’
Domain analysis
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Top model info
Secondary structure/disorder
Domain analysis
Detailed template information
Example results
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Main results table
Actual Model!Not just a picture of the template –click to download model
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How accurate is my model?• Simple question with a complicated answer!• RMSD very commonly used, but often misleading• Modelling community uses TM score for benchmarking: essentially the percentage of alpha carbons superposable on the answer within 3.5Å. Prediction of TM‐score coming soon.
• Focused on the protein core, rather than loops and sidechains.
Interpreting results
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• MAIN POINT: The confidence estimate provided by Phyre2 is NOT a direct indication of model quality – though it is related…
• It is a measure of the likelihood of homology• Model quality can now be assessed using the new Phyre Investigator (more later)
• New measure of model quality coming soon..
Interpreting results
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Sequence identity and model accuracy • High confidence (>90%) and High seq. id. (>35%): almost always very accurate: TM score>0.7, RMSD 1‐3Å
• High confidence (>90%) and low seq. id. (<30%) almost certainly the correct fold, accurate in the core (2‐4Å) but may show substantial deviations in loops and non‐core regions.
Interpreting results
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Interpreting results
100% confidence, 56% sequence identity, TM‐score 0.9
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Interpreting results
100% confidence, 24% sequence identity, TM‐score 0.8
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Checklist
• Look at confidence• Given multiple high confidence hits, look at % sequence identity
• Biological knowledge relating function of template to sequence of interest
• Structural superpositions to compare models –many similar models increase confidence
• Examine sequence alignment
Interpreting results
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Main results table
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Alignment view
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Alignment view
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Alignment view
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Checklist
• Secondary structure matches• Gaps in SS elements indicate potentially wrong alignment
• Active sites present in the Catalytic Site Atlas (CSA) for the template highlighted – look for identity or conservative mutations when transferring function
• Alignment confidence per residue
Alignment interpretation
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• The STRUCTURAL effects of point mutations on structure will NOT be modelled accurately
Checklist• Is it near the active site? • Is it a change in the hydrophobic core? • Is it near a known binding site? (can predict with e.g. 3DLigandSite)
• Phyre Investigator can help (see later)
Mutations
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All depends on your purpose.
• Good enough for drug design? – probably if the sequence identity is very high (>50%)
• Sometimes good enough if far lower seq id but accurate around site of interest.
• High confidence but low seq i.d. still very likely correct fold, useful for a range of tasks.
Is my model good enough?
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• “Normal” Mode• “Intensive” Mode• Advanced functions
How does Phyre2 work?
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• Individual domains in multi‐dom proteins often modelled separately
• Regions with no detectable homology to known structure unmodelled
• Does not use multiple templates which, when combined could result in better coverage
Shortcomings of ‘normal’ Mode
Thus need a system to fold a protein without templatesand combine templates when we have them
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structure simplification
Protein backbone
Small hydrophilic sidechain
Large hydrophobic sidechain
BackboneC‐alpha
Poing – simplified folding model
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ARNDLSLDLVCS……. HMM
PSI‐Blast
Hidden Markov Model DB of KNOWN
STRUCTURES
Extract pairwisedistance constraints
POING: Synthesise from virtual ribosome.Springs for constraints. Ab initio modellingof missing regions.
FINAL MODELHMM‐HMM matching
Phyre + Poing
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Intensive mode
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• Designed to handle mutliple domains or proteins with substantial stretches of sequence without detectable homologous structures.
• POOR at ab initio regions• GOOD at combining multiple templates covering different regions
Intensive mode
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• Relative domain orientation will NOT generally be correct if those domains come from different PDB’s with little structural overlap.
Intensive mode
✔Query
Template 1Template 2
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• Relative domain orientation will NOT generally be correct if those domains come from different PDB’s with little structural overlap.
Intensive mode
✖
?
Query
Template 1Template 2
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“Intensive” does not always equal “Better”!
Checklist• Always use normal mode first to understand what regions can be well modelled
• Multiple overlapping high confidence domains? Good, try intensive. Otherwise skip it.
• Danger of “spaghettification”• Active development, new version ‘soon’
Intensive mode
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• “Normal” Mode• “Intensive” Mode• Advanced functions
– Phyre Investigator on web page including mutational analysis by SuSPect
– Log in to use expert mode
How does Phyre2 work?
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Phyre Investigator
• What parts of a model are reliable?• What parts may be functionally important? (guide mutagenesis, understand mutants/SNPs)
• What residues are involved in interactions with other proteins?
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• Clashes• Rotamer outliers• Ramachandran outliers• ProQ2 model quality assessment• Alignment confidence (HHsearch)• Conservation/evolutionary trace (Jenson‐Shannon divergence
–far faster and just as accurate as ET)• Catalytic Site Atlas• Disorder• Pocket detection (Fpocket)• Protein interface residues (PI‐Site, ProtinDB)• Conserved Domain Database ‘conserved features’ for NCBI‐
curated domains
Phyre Investigator
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• Will a SNP effect my protein’s function?• New method: SuSPect by Chris Yates• Integrated into Phyre Investigator• Also standalone serverYates CM, Filippis I, Kelley LA, Sternberg MJE. SuSPect: Enhanced Prediction of Single Amino Acid Variant (SAV) Phenotype Using Network Features. Journal of Molecular Biology. 2014;426(14):2692‐2701.
Effect of Mutations?
Phyre Investigator
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Phyre Investigator
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Phyre Investigator
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Phyre Investigator
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SuSPect – Phenotypic effect of amino acid variants
Sequence conservation•PSSM•Pfam domain•Jensen‐Shannon entropy
Structural features•Predicted solvent accessibility
Network features•Protein‐protein interaction (PPI) as domain centrality
Domain
Conserva on
Secondary structure
Solvent accessibility
Intrinsic disorder
Interactome
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SuSPect – Results on non-training data (VariBench)
1 − Specificity
Sen
sitiv
ity
0.0
0.2
0.4
0.6
0.8
1.0
0.0 0.2 0.4 0.6 0.8 1.0
SuSPectCondelPolyPhen−2SIFTMutationAssessorFATHMM
Specificity TPTP TN
Sensitivity TPTP FP
SuSPectMutationAssessor
SIFT
1 ‐ Specificity
PolyPhen2
Benchmark consists of 20k SNPs (15k Neutral, 5k pathogenic)
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Neonatal diabetes
• Arg 201 His in ATP‐sensitive inward rectifier potassium channel 11 (Kir6.6)
• SuSPect gives score of 87/100 – high probability of diseaseassociated
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Phyre2 yields model which suggest structural basis for disease
Arg 201 forms H‐bond with main chain O
His in variant could not form similar interaction
Most variants predict to be disease associated
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Advanced functions
Register and Log in to access Expert Mode
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• PhyreAlarm – automatically re‐run tricky sequences every week
• BackPhyre – compare a structure to up to 30 genomes
• One‐To‐One Threading – use specfic PDB for model building
• Batch Jobs – run many sequences at once
• Job Manager – keep track of your jobs and history
Advanced functions
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• PhyreAlarm – automatically re‐run tricky sequences every week
• BackPhyre – compare a structure to up to 30 genomes
• One‐To‐One Threading – use specfic PDB for model building
• Batch Jobs – run many sequences at once
• Job Manager – keep track of your jobs and history
Advanced functions
![Page 72: Protein structure prediction using Phyre2 and ... · The Phyre2 web portal for protein modeling, prediction and analysis Kelley,LA, Mezulis S, Yates CM, Wass MN &Sternberg MJES Nature](https://reader036.fdocuments.us/reader036/viewer/2022081611/5f0499067e708231d40ec2d7/html5/thumbnails/72.jpg)
• Sometimes no confident homology detected• Automatically try every week as new structures are deposited in the PDB
• Receive an email if hit found• PhyreAlarm auto‐suggested in cases where sequence has low coverage by confident hits
• Two clicks adds your sequence to the alarm queue
PhyreAlarm
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SVYDAAAQLTADVKKD…….
PhyreAlarm
HMM
Newly added structure HMMs
HMM‐HMM matching
User sequence
Confident hit?
Newly solved PDB Structures added WEEKLY
Yes
No
Try again next week
Perform full Phyre
modellingEmail resultsNew 3D model
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• PhyreAlarm – automatically re‐run tricky sequences every week
• BackPhyre – compare a structure to up to 30 genomes
• One‐To‐One Threading – use specfic PDB for model building
• Batch Jobs – run many sequences at once
• Job Manager – keep track of your jobs and history
Advanced functions
![Page 75: Protein structure prediction using Phyre2 and ... · The Phyre2 web portal for protein modeling, prediction and analysis Kelley,LA, Mezulis S, Yates CM, Wass MN &Sternberg MJES Nature](https://reader036.fdocuments.us/reader036/viewer/2022081611/5f0499067e708231d40ec2d7/html5/thumbnails/75.jpg)
• Does a structure I’m interested in exist in an organism?
• 30 searchable genomes to‐date. • Scan multiple genomes at a time. Quite fast.• New version will allow users to upload their own genomes of interest.
BackPhyre
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SVYDAAAQLTADVKKDLRDSWKVIGSDKKGNGVALMTTLFADNQETIGYFKRLGNVSQGMANDKLRGHSITLMYALQNFIDQLDNPDSLDLVCS…….
BackPhyre
HMM
Hidden Markov
Model DB of Genomes
HMM‐HMM matching
User structure
Rank Hit Confid‐ence
1 Gi…
2 Gi..
3 Gi..
. .
. .
Ranked list of genome hits
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• PhyreAlarm – automatically re‐run tricky sequences every week
• BackPhyre – compare a structure to up to 30 genomes
• One‐To‐One Threading – use specfic PDB for model building
• Batch Jobs – run many sequences at once
• Job Manager – keep track of your jobs and history
Advanced functions
![Page 78: Protein structure prediction using Phyre2 and ... · The Phyre2 web portal for protein modeling, prediction and analysis Kelley,LA, Mezulis S, Yates CM, Wass MN &Sternberg MJES Nature](https://reader036.fdocuments.us/reader036/viewer/2022081611/5f0499067e708231d40ec2d7/html5/thumbnails/78.jpg)
• Useful if you:a) Know a better template than found by Phyre2b) Have your own structure not yet in the PDBc) Model a a lower‐ranked (>20) template d) Want more expert control over alignment options: local/global, secondary structure weight etc.
One-to-One Threading
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SVYDAAAQLTADVKKDLRDSWDLVCS…….
One to one threading
HMM ofUser
structure
HMM‐HMM matching
User structure
KLRGHSITLMYALQNNPDSLDLVCS…….
User sequence HMM ofuser
sequence
Final model
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Future
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PhyreStorm
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• Searching Topology with Rapid Matching• Structural search and alignment of the entire PDB in under 1 minute.
• Go directly from a Phyre2 model and find all other similar structures rapidly.
• Beta released
PhyreStorm
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PhaserPhyre
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PhyreRisk (with Prof R Houlston ICR)
Integrate disease networks, SNPs, GWAS, protein structure and complexes
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PhyreRisk
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Protein structure prediction using Phyre2
and understanding genetic variants.Prof. Michael SternbergDr. Lawrence KelleyMr. Stefans Mezulis
Dr Chris Yates
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Today
• 10.00 – 11.00 Lecture• 11.00 – 11.30 Tea/Coffee• Courtyard, West Medical Building• 11.30 – 1.00 Hands on workshop using
Phyre2
• Computer Cluster 515, West Medical Building
Many thanks to Glasgow Polyomicsand Amy Cattanach
Timetable