52925 proteiinianalyysin ty t how hands on workshop on protein analysis l.
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52925 Proteiinianalyysin työt HOW – hands-on workshop on protein analysis Liisa Holm Instructors Patrik Koskinen Samuli Eldfors Xuan Hung Ta Martin Heger Petri Törönen Jussi Nokso-Koivisto Course web page http://ekhidna.biocenter.helsinki.fi/how Schedule Talks Exercises

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instructors
Instructors
  • Patrik Koskinen
  • Samuli Eldfors
  • Xuan Hung Ta
  • Martin Heger
  • Petri Törönen
  • Jussi Nokso-Koivisto
course web page
Course web page

http://ekhidna.biocenter.helsinki.fi/how

  • Schedule
  • Talks
  • Exercises
  • Course assignments
  • Instructions for computer use
topics
Week I

Monday

Introduction

Pairwise alignments

Tuesday

Manual editing of sequence alignment

Wednesday

Secondary structure prediction

Thursday

Structure visualisation

Friday

Comparative modelling

Week II

Monday

Phylogenomics

Tuesday

Sequence classifications

Wednesday

Protein-protein interactions

Thursday

Structure classifications

Friday

Review day

Week III

Work on course assignments

Topics
course organization
Course organization
  • 1st and 2nd week (Structured)
    • Demonstrations (12 -…)
    • Practical exercises (…-17)
  • 3rd week (Self-organized)
    • Course assignment
      • Instructor available two hours daily
    • Discussion on Tuesday 13-15
  • Written report due 8th December
mode of work
Mode of work
  • Demonstrations
  • Practical exercises
    • Structured questions
    • You should first try yourself, then ask team mate, then ask instructor
    • Discuss results with team mate
  • Course assignments
    • Written reports, due 8 December
    • Two sequence assignments per team
    • Course grade based on report
objectives
Objectives
  • Infer function and/or structure starting from the amino acid sequence of a query protein
    • Identify related sequences, place in family
    • Identify conserved positions in sequence and structure
  • Learn to use representative web-based tools
  • No programming, no Unix/Linux
introduction
Introduction
  • Most cellular functions are performed or facilitated by proteins.
    • Primary biocatalyst
    • Cofactor transport/storage
    • Mechanical motion/support
    • Immune protection
    • Control of growth/differentiation
linear dna
Linear DNA

Watson & Crick (1953)

3d structure
3D structure

Myoglobin

Kendrew & Perutz (1957)

1mbn

evolution sequence structure function
EvolutionSequence – Structure - Function

Natural selection

DNA sequence

Protein function

Protein sequence

Protein structure

what can sequence analysis do
What can sequence analysis do?
  • Homology
    • Inference of inherited complex features: what is conserved is important
    • Most powerful approach
    • Good tertiary structure prediction
  • Diagnostic patterns
    • E.g. subcellular localization signals
  • Physical preferences
    • Good secondary structure prediction
    • Prediction of transmembrane segments
    • Poor ab initio tertiary structure prediction
application finding homologues
Application:Finding Homologues
  • Find Similar Ones in Different Organisms
  • Human vs. Mouse vs. Yeast
    • Easier to do Expts. on latter!

(Section from NCBI Disease Genes Database Reproduced Below.)

Best Sequence Similarity Matches to Date Between Positionally Cloned

Human Genes and S. cerevisiae Proteins

Human Disease MIM # Human GenBank BLASTX Yeast GenBank Yeast Gene

Gene Acc# for P-value Gene Acc# for Description

Human cDNA Yeast cDNA

Hereditary Non-polyposis Colon Cancer 120436 MSH2 U03911 9.2e-261 MSH2 M84170 DNA repair protein

Hereditary Non-polyposis Colon Cancer 120436 MLH1 U07418 6.3e-196 MLH1 U07187 DNA repair protein

Cystic Fibrosis 219700 CFTR M28668 1.3e-167 YCF1 L35237 Metal resistance protein

Wilson Disease 277900 WND U11700 5.9e-161 CCC2 L36317 Probable copper transporter

Glycerol Kinase Deficiency 307030 GK L13943 1.8e-129 GUT1 X69049 Glycerol kinase

Bloom Syndrome 210900 BLM U39817 2.6e-119 SGS1 U22341 Helicase

Adrenoleukodystrophy, X-linked 300100 ALD Z21876 3.4e-107 PXA1 U17065 Peroxisomal ABC transporter

Ataxia Telangiectasia 208900 ATM U26455 2.8e-90 TEL1 U31331 PI3 kinase

Amyotrophic Lateral Sclerosis 105400 SOD1 K00065 2.0e-58 SOD1 J03279 Superoxide dismutase

Myotonic Dystrophy 160900 DM L19268 5.4e-53 YPK1 M21307 Serine/threonine protein kinase

Lowe Syndrome 309000 OCRL M88162 1.2e-47 YIL002C Z47047 Putative IPP-5-phosphatase

Neurofibromatosis, Type 1 162200 NF1 M89914 2.0e-46 IRA2 M33779 Inhibitory regulator protein

Choroideremia 303100 CHM X78121 2.1e-42 GDI1 S69371 GDP dissociation inhibitor

Diastrophic Dysplasia 222600 DTD U14528 7.2e-38 SUL1 X82013 Sulfate permease

Lissencephaly 247200 LIS1 L13385 1.7e-34 MET30 L26505 Methionine metabolism

Thomsen Disease 160800 CLC1 Z25884 7.9e-31 GEF1 Z23117 Voltage-gated chloride channel

Wilms Tumor 194070 WT1 X51630 1.1e-20 FZF1 X67787 Sulphite resistance protein

Achondroplasia 100800 FGFR3 M58051 2.0e-18 IPL1 U07163 Serine/threoinine protein kinase

Menkes Syndrome 309400 MNK X69208 2.1e-17 CCC2 L36317 Probable copper transporter

what you will learn
What you will learn
  • Multiple alignment
    • Used as input to many prediction tools
    • Improves sequence-structure alignment
    • Identify functional sites
  • Protein structure
    • Visualisation
    • Comparative modelling
  • Using phylogeny in function assignment
    • Family classifications
flowchart

Query = Protein sequence

Sequence similarity to other proteins?

Yes: does similarity imply homology?

Yes: place query in family tree

Known function(s) in family?

Yes

Transfer function

Verify conservation of functional motifs

No

Motif search

Use other data

Known structure in family?

Yes

Comparative modelling

Validate motifs against 3D model

No

Secondary structure prediction

No: use single sequence methods

No: single sequence methods

Motif search

Secondary structure prediction

Use other data

Flowchart
course assignments
Course assignments
  • Goal: using the flowchart, what can you say, with what confidence, about the structure and function of the protein?
  • Max length of report ~10 pages. No need to show negative results.
  • More detailed guidelines given on Day 10.
teams
Team n, n=1,…,12, works on both sequence_nA and sequence_nB

A and B sequences have been selected to present different challenges, therefore it is strongly recommended that the team members work together on both sequences

Sequences are here: http://ekhidna.biocenter.helsinki.fi/how/proteinlist.fasta

Teams
preparing sequence reports
Preparing sequence reports
  • Week 3 is reserved for preparing the reports.
    • Experience has shown that students progress at different speeds. Fast students may try the tools out on their sequence assignments during weeks 1-2.
    • Checkpoint on Tuesday (Day 12)
      • It is expected that sequence database searches and some downstream analyses have been done by then
      • The purpose is to summarize progress and discuss strategies forward